Abstract Book All

SMAP Conference, September 19th-22nd 2011, Avignon
1/381
Popes' Palace photos credit : Yann Fareins / Noir d'Ivoire
2/381
Table of contents
General information.....................................................................5
SMAP 2011..................................................................................................6 Avignon.......................................................................................................6 Organizing committee.................................................................................8 Scientific committee....................................................................................9 Sponsorship...............................................................................................10 Popes' palace plan.....................................................................................12 Practical information.................................................................................14 Oral sessions organisation........................................................................16 Poster sessions organisation.....................................................................17 Exhibitor List..............................................................................................18 Exhibition organisation.............................................................................19 Program.....................................................................................................21 Invited conference.....................................................................................27 Selected short oral communications.........................................................47 Posters.......................................................................................................87
Abstracts.....................................................................................27
Participants list.........................................................................357 Author Index.............................................................................369
3/381
4/381
General information
5/381
SMAP 2011
We are delighted to welcome you to the new SMAP (Mass Spectrometry and Proteomic Analyses) scientific meeting, which will take place in Avignon, France, from September 19 th to 22nd, 2011. This scientific congress is co-organized by SFSM (Socit Franaise de Spectromtrie de Masse) and SFEAP (Socit Franaise d'Electrophorse et d'Analyse Protomique). The SMAP2011 congress has for objective to promote scientific disciplines called to play crucial roles in the XXIth century biology and chemistry. The whole French-speaking scientific community will exchange in Avignon on federative themes, subjects in emergence and new technological developments domains related to mass spectrometry and proteomics.
Avignon
Avignon is one of the most beautiful and famous cities in France and well-known around the world thanks to the famous song about the bridge of Avignon. Its beautiful and typically medieval city center on both banks of the Rhne river attracts millions of tourists each year. Named the City of the Popes or Altera Roma, Avignon retains the indelible mark of the Popes stay in the city, which was for a while the capital of the Medieval western world. Today, it is a prestigious cultural capital with its world-renowned Theatre Festival. Within a short time, the tourist can visit the most various sites: Pont du Gard, Nmes, Arles, the Camargue, Luberon and Ventoux, Baux de Provence, St Rmy and the Alpilles are close to the town. The variety of attractions and sites to visit can enrich your trip to Provence. The era of the Popes somewhat eclipses other events in what is a long and tumultuous history. At the crossroads of the big trade and migratory routes between northern and southern Europe and between Italy and Spain, the city played a major role in European history. A Phoenician trading post during the High Antiquity, Avignon then became a flourishing Roman town. It suffered greatly from the barbarian invasions, followed by those of the Moors and the Francs in the High Middle Ages. With the expansion of trade, and benefiting from its strategic position and its bridge over the Rhne, it had the status of a free town, strong and arrogant enough to defy the King of France. The presence of the Popes made Avignon the capital of the Medieval western world in the 14th century. A papal territory up until the French Revolution, the city actually benefited little from the first Industrial Revolution. It entered into relative anonymity in the 19th century only to come back as a cultural capital in the 20th century. Avignon is the cradle of the Flibrige, a revival of Provenal literature and its Theatre Festival, started in 1946 by Jean Vilar, gives it international prestige. During the 14th century, the city of Avignon was part of the Pontifical States, and as such was sheltered from war and destruction. Since the High Middle Ages, Avignon has been a vital intellectual and commercial cross-road in Europe, a choice location for exchange, meetings and discussions. Today, its internationally-known cultural influence and attraction have made it one
6/381
of the most prestigious conference sites in the world. The International Congress Center was created in 1976 within the outstanding premises of the Palace of the Popes and hosts many events throughout the entire year. The Congress Center, designed for conventions, seminars, and meetings, now occupies two wings of the Popes Palace. The Jeanne Laurent Building, an outlying building just outside the Popes Palace Congress Center, is located in the former papal gardens and can host working meals or exhibits relating to congresses held in the Congress Center. The Jeanne Laurent building is made up of four vaulted rooms in exposed stone, with magnificent views over the Rhne river, offering a splendid panorama of the Rhne Valley.
7/381
Organizing committee
Jean Armengaud1 Alain Dedieu1 Christian Godon1 Vronique Malard1 Olivier Pible1 Jean-Charles Gaillard1 Philippe Gurin1 Cline Bland1 Batrice Alonso1 Anne-Hlne Davin1 Nicole Sage1 Jrme Garin2 Christophe Bruley2 Myriam Ferro2 Damien Barthe2 Vronique Dupierris2 Annie Adrait2 Alexandra Kraut2 Sabine Brugire2 Cline Fleury2 Marie Arlotto2 Mathieu Baudet2 Yves Vandenbrouck2
Laboratoire de biochimie des systmes perturbs (LBSP), Institut de biologie environnementale et biotechnologie (IBEB) Direction des sciences du vivant (DSV) Commissariat lEnergie Atomique et aux Energies Alternatives (CEA), BP17171, 30207 Bagnols-sur-ceze
Etude de la Dynamique des Protomes (EDyP) Laboratoire Biologie Grande Echelle (BGE) U1038 INSERM/CEA/UJF Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV) Commissariat lEnergie Atomique et aux Energies Alternatives (CEA),17 rue des Martyrs, 38054 Grenoble
8/381
Scientific committee
Marc Bonneu, Plateforme Proteome, Centre de Gnomique Fonctionnelle Bordeaux, 146 rue
Lo Saignat, F-33076 Bordeaux Cedex
Julia Chamot-Rooke, Laboratoire des Mcanismes Ractionnels, CNRS UMR 7651, Ecole
Polytechnique, 91128 Palaiseau Cedex
Philippe Dugourd, Laboratoire de Spectromtrie Ionique et Molculaire, UMR5579

CNRS/Universit Lyon 1, 43 bd du 11 Novembre 1918, F-69622 Villeurbanne Cedex
Jrme Garin, Laboratoire Biologie Grande Echelle (BGE), U1038 INSERM/CEA/UJF, CEA), 17
rue des Martyrs, F-38054 Grenoble Cedex,
Bruno le Bizec, LABERCA - U1329 INRA, Ecole Nationale Vtrinaire, Agroalimentaire et de

lAlimentation Nantes Atlantique (ONIRIS), Atlanple Site de la Chantrerie - BP 50707, F-44307 NANTES Cedex 3
Philippe Marin, Institut de Gnomique Fonctionnelle, CNRS UMR 5203, INSERM U661,
Universits Montpellier I & II, 141, rue de la Cardonille, F-34094 Montpellier Cedex 5
Charles Pineau, INSERM U625, Proteomics Core Facility Biogenouest, Campus de Beaulieu,
Universit de Rennes I, F-35042 Rennes, France
Michel Salzet, Laboratoire de Spectromtrie de Masse Biologique Fondamentale et Applique

(FABMS), EA 4550, IFR 147, Cit Scientifique, Universit Lille Nord de France, Lille 1, 59650 Villeneuve d'Ascq
9/381
Sponsorship
10/381
11/381
Popes' palace plan
1-Salles des Gardes : Welcome / Informations / Registration 2-Cellier Benoit XII : Oral conferences (even number) & Manufacturer conferences 3-Paneterie : Poster sessions n1 & n2 5-Conclave : Plenary conferences & Oral conferences (odd number) 7-Chambre du trsorier : Manufacturer conferences 9-Herses Champeaux : SFEAP & SFSM Young 12-Grande audience : Exhibitors room & Coffee breaks 13-Espace Jeanne Laurent : Lunch & Gala dinner
12/381
1-Salles des Gardes : Welcome /

Informations / Registration
2-Cellier Benoit XII : Oral conferences (even number) & Manufacturer conferences
3-Paneterie : Poster sessions n1 & n2
5-Conclave : Plenary conferences &Oral

conferences (odd number)
7-Chambre du trsorier : Manufacturer
12-Grande audience : Exhibitors room &

Coffee breaks
conferences
13/381
Practical information
Office hour of desks Please collect your satchel, program book, name badge from the registration desk. The registration desk will open at the Salle des Gardes : Monday 19 September, 2 pm - 7 pm Tuesday 20 September, 8 am - 6:30 pm Wednesday 21 September, 8 am - 6:30 pm Thursday 22 September, 8 am - 4 pm Official language The official languages of the congress are English and French according to the laws of the French republic on the use of the French language. The oral communications and the posters can be in French, in that case, their summary and title are presented in French in the program and the book of summaries. Staff Voluntaries will be at your disposal to welcome you, guide you in the center of the Popes' Palace Conference Center, during the sessions and the excursions. Do not hesitate to seek them, they are recognizable in their T-shirt with the logo of the SMAP2011 Avignon congress. Badges For security and regulation reasons, please wear your name badge at all times. It is your admission to all sessions, breaks in the "Grande Audience" room, lunches in Jeanne Laurent Building. Cellular phones Cellular phones must be switched off in the conference rooms. Lost and Found For lost and found personal belongings, please contact the information desk in the Salle des Gardes. First Aid In case of emergency, please, contact the information desk in the Salle des Gardes. Hotline Under this phone number you can reach the desk at all office hours : +33 490 27 51 50. Contacts Please feel welcome to provide the Local Organizing Committee with any feedback or to ask for clarification of any information. Registration queries, please contact Ms. Cline Gallard by email c.gallard@palaisdes-papes.com or phone +33 490 27 50 57 Scientific queries, please contact Mr Jean Armengaud or Mr Jrme Garin by email, respectively jean.armengaud@cea.fr and jerome.garin@cea.fr. Smoking Please remember that smoking is not allowed in the congress center. Wifi Wireless connexion will be available in Grande audience and Salle des gardes rooms.
Coffee breaks Morning and afternoon coffee breaks will be served only in the Grande Audience room. Changing room Participants are not allowed to carry out drinks A changing room will be open throughout the conference in the main entrance near the or food in working rooms. Salle des gardes. It will be possible to store luggage in this changing room area. Banks Please, note that there will be no exchange facilities at the International Conference Toilets Center. Lots of banks are present in Avignon They are situated near the Salle des Gardes, center all around the PopesPalace Conference between the Paneterie and The Conclave hall, in Jeanne Laurent Building and near the Center. Grande Audience.
14/381
15/381
Oral sessions organisation

They will occur in the room of Conclave and Cellier Benoit XII (respectively n5 and n2 on the plan page 12).
Instructions for oral communication

Short oral presentations will be strictly limited to 15 minutes. A 5-minute period
between presentations will accommodate questions, discussion, and introduction of the next speaker. This 5-minute period belongs to the audience and to the chairs of the session, not to the speakers. TIME LIMITS WILL BE STRICTLY ENFORCED BY SESSION CHAIRS. Microsoft PowerPoint and Adobe Acrobat Reader are the only acceptable audiovisual formats for electronic presentations. Accepted formats are: .ppt, .pps or .pdf. The venue will be equipped with a dedicated PC laptop operated under windows. Please, bring your presentation on a Flash drive. Uploading your presentation should be done preferentially once you register for the SMAP meeting upon your arrival in Salles des Gardes. You should upload your presentation either the day before for an oral presentation in the morning or in the morning if your presentation is scheduled in the afternoon. Please, be present in the conference room 15 min before the beginning of your session in order to have time to discuss with the session chair that will be in charge to introduce you. We recommend to all speakers to have all their slides in English as a courtesy for our foreign guests. Oral presentation of your talk in English will be appreciated.
Organisation des confrences

Elles se drouleront dans les salles du Conclave et Cellier Benoit XII (respectivement n5 et n2 sur le plan page 12).
Instructions pour les confrences slectionnes
Dure des prsentations : 15 min prcisment et 5 min gres par le modrateur
permettant des questions / discussions et lintroduction de lorateur suivant. Compte tenu de la configuration du centre des congrs et de la circulation entre les salles en session parallle, les horaires seront strictement respects. Fichier : Les prsentations doivent tre faites sous Powerpoint ou Adobe Acrobat Reader. Fichiers accepts : .ppt, .pps ou .pdf. Le PC de projection sera sous Windows. Apportez votre prsentation sur cl USB. Remise des fichiers : ils devront tre remis de prfrence au moment de votre enregistrement laccueil du palais des Papes (Salle des Gardes). En dernier recours : la veille si votre confrence est le matin ; le matin si votre confrence est laprs-midi. Etre prsent 15 min avant le dbut de votre session dans la salle o se droulera celleci afin de rencontrer le modrateur en charge dintroduire votre prsentation. Nous recommandons tous les confrenciers de prsenter lensemble de leurs diapositives en anglais par courtoisie auprs de nos invits anglophones. La prsentation orale de vos travaux en anglais serait trs apprcie.
16/381
Poster sessions organisation

There will be two poster sessions, both will be in the Paneterie room (n3 on the plan, page 12). Session n1 : Poster P1 to P134 Session n2 : Poster P135 to P268 Display : poster should be mounted : Session n1 : From tuesday 20 september before 8:30am, till wednesday 21 september 12:00am. At the end of session n1, posters will be removed to give way to those of session n2, Session n2 : From wednesday 21 september before 1:30pm till thursday 22 september 3:30pm.
Organisation sessions posters
Il y aura deux sessions poster, les deux se tiendront dans la salle Paneterie (n3 sur le plan page 12). Session n1 : Poster P1 P134 Session n2 : Poster P135 P268 Affichage : votre poster devra tre mis en place : Session n1 : Le mardi 20 septembre avant 8h30 jusqu'au mercredi 21 septembre 12h00. A la fin de la session n1 les posters seront enlevs pour faire place ceux de la session n2. Session n2 : Le mercredi 21 septembre avant 13h30 jusqu'au jeudi 22 septembre 15h30.
17/381
Exhibitor List
1 - AB SCIEX 2 - ADVION
Mr ees VLAK Mr Jean- Baptiste VINCENDET
19 - LABTECH
Mr Lionel Chauvin
20 - LECO FRANCE
3 - AGILENT TECHNOLOGIES
Mr Frdric METRAL
Mr Sergio MELIS Mr Guillaume CHARLOT
21 - LGS LAB GAZ SYSTEMS

Mr Patrick VESCOVI
4 - ASA - ADVANCED SOLUTIONS ACCELERATOR

Mr Valentin BOUQUET Mr Frdric VIART
22 - MS VISION BV
Mr Gerard VAN DER LAAN Mr Nico WORTEL
5 - BIO RAD
23 - NONLINEAR DYNAMICS
Dr Andy BORTHWICK Mrs Agns CORBIN
Mr Patrick BELLEMIN Mr Stphane FESSEMAZ
6 - BIOSOLVE CHIMIE
Mr Julien GUERIN
24 - PERKINELMER
7 - BRUKER BECKMANN
Mrs Chirstine HERR
Mrs Karima BAUDIN Mr Christophe CLARYSSE
25 - PROMEGA
8 - PROTEINSIMPLE (CELL BIOSCIENCES)

Mr Thierry SALOMON
Mr Emmanuel CRENN
26 - PROTEABIO EUROPE
Mrs Christine AYOUB Mr Alban LECOLLEN Ms Jana LEE
9 - CEM WAVES
Mr Nicolas RAYNAL
10 - CHROMACIM SAS
27 - PROTEOME SOFTWARE 28 - SERVA ELECTROPHORESIS GMBH

Mr Philippe BOGARD Dr Reiner WESTERMEIER
Mr Pierre BERNARD SAVARY
11 - CLUZEAU INFO LABO

Mrs Catherine BALTHASAR
12 - COVALX
Mr Alexis NAZABAL
29 - SHIMADZU
13 - DECODON GMBH
Mr Markus KOLBE Dr Martin LINKE
Mr Mikael LEVI Mr Stphane MOREAU
30 - TECAN FRANCE SAS

Mr David RODARIE Mr Xavier SAUNIER
14 - DIONEX
Mr Claude NETTER
31 - THERMO ELECTRON SAS

Mr Christophe DABADIE Mr Louis DESCHANEL Mr Jocelyn DUPUY Mr Thierry LEGOUPIL Mr Etienne MAOUT Mrs Madeleine PEYTAVIN
15 - EURISO-TOP
Mr Jean- Louis SCHAFFAR Mr Gatan SEEBURN
16 - F-DBS
Mrs Fabienne PALGE
17 - GENGAZ EURL
Mr Eric LEPOUTRE
32 - WATERS
Mrs Sophie BERTAUX
18 - JEOL EUROPE
Mr Akihiko KUSAI Mr Jean- Pierre MUNIER
18/381
Exhibition organisation
In Grande Audience room (n12 on plan page 12)
19/381
20/381
Program
- Codes in square brackets are the abstracts' identifiers : [Ix] are for Invited conferences [Ox] are for selected Oral conferences - Numbers in brackets next to room name refering to popes' palace plan, page 12.
Monday September 19th

9:00am-12:00am 1:30pm-3:30pm 1:30pm-3:30pm 1:30pm-3:30pm 4:00pm-4:30pm 4:30pm-5:30pm Petite Cuisine (16) Salle des gardes (1) Training day Imagerie MALDI-TOF : des drogues aux protines Conferee check-in
Herses-Champeaux (9) SFEAP & SFSM Young SFSM & SFEAP presidents' welcome word Opening conference 1 Graham COOKS Ambient Ionization and Miniature Mass Spectrometers [I7] Opening conference 2 Michel DESJARDINS Proteomics analyzes reveal unexpected aspects of the evolution of phagosomes over 1.2 billion years [I8] Grande Audience (12) Cocktail & Inauguration Exposition Private guided tour of the Popes' Palace
Conclave (5) 5:30pm-6:30pm
6:30pm-9:00pm 7:00pm-9:00pm
21/381
Tuesday September 20th

8:30am-9:25am Conclave (5) Plenary conference 1 - Anne-Claude GAVIN - A systematic screen for proteinlipid interactions in Saccharomyces cerevisiae [I10] Session 1 - System's Biology Paola PICOTTI - Targeted proteomics from proteins to proteome maps : potential and bottlenecks [I16] Rgis Lavigne - Systems analysis fermentation and respiration [O25] Break : Poster session n1 Coffee & Exposition Hans C. Hrlimann - Using proteomics to identify AICAR cellular targets [O27] Conclave (5) 12:10am 9:35am-12:30am 09:35am 10:10am Cellier Benoit XII (2) Grmy Clair - System biology insights into the global remodeling of proteome and pathogenicity of Bacillus cereus induced by oxydoreduction potential [O32] Bertrand Fabre - Characterization of cellular proteasome complexes diversity by proteomic approaches [O13] Session 2 - Imaging & Mobility Jennifer BRODBELT - Development and Applications of Photodissociation for Biological Applications [I4] Grgory Hamm - Dveloppement Mthodologique pour la Quantification en Imagerie par Spectromtrie de Masse (qMSI) [O26] Break : Poster session n1 Coffee & Exposition Daniel Lafitte - Quelle voie pour la caractrisation d isoformes protiques sur coupe de tissu par MALDI in source decay ? [O31] Cellier Benoit XII (2) Claudia Bich - Compatibilit entre histologie et imagerie par spectromtrie de masse ToF-SIMS : dtection et cartographie de lipides au sein de tissus biologiques [O5] Patrice Garcia - Dtection de lepo humaine recombinante dans les fluides biologiques equins par LC-MS/MS [O34] Jeanne Laurent (13) Cellier Benoit XII (2) Lunch Manufacturer conference : Agilent of budding yeast
9:35am-12:30am 9:35am 10:10am 10:30am Conclave (5)
Paneterie (3) Grde Audience (12)
11:30am 11:50am
10:30am Paneterie (3) Grde Audience (12) 11:30am
11:50am
12:10 12:40am-1:55pm 12:55am-2:25pm 1:40pm-2:25pm 2:00pm-2:25pm
Chbr du Trsorier (7) Manufacturer conference : AB Sciex Grde Audience (12) Exposition
22/381
Tuesday September 20th

2:30pm-3:25pm 3:35pm-6:30pm 3:35pm 4:10pm Conclave (5) Conclave (5) Plenary conference 2 - Martin JARROLD - Charge Detection Mass Spectrometry [I12] Session 3 - Instrumentation Perdita BARRAN - Ion Mobility Mass Spectrometry for Intrinsically Disordered Proteins a SWOT analysis [I2] Quentin Enjalbert - Photo-SRM: laser photo-dissociation improves detection selectivity of selected reaction monitoring mode [O7] Break : Poster session n1 Coffee & Exposition Mathieu Dupr - LDI-MS of proteolytic synthetic model peptides deposited on silicon-based nanowires: an alternative to MALDI PMF? [O17] Conclave (5) David Touboul - ETD pression atmosphrique [O6] Marion Girod - Optical profiling of an Agilent Jet Stream Technology electrospray by Laser-Induced-Fluorescence coupled to mass spectrometry measurements [O14] Session 4 Proteome Dynamics Christopher OVERALL - Traveling to the Ends of the Proteome World. Positional N-terminal and C-terminal proteomics deciphers protein terminal and proteolytic post-translational modifications in the HPP [I15] Catherine Moali - Use of quantitative proteomics to study function and specificity of an emerging protease family in cellbased assays [O22] Paneterie (3) Grde Audience (12) 5:30pm Break : Poster session n1 Coffee & Exposition Anne Gonzalez de Peredo - Evaluation of label-free quantitative proteomic methods together with sample fractionation for the large-scale analysis of inflammatory endothelial cells [O18] Cellier Benoit XII (2) Joseph Gault - Post Translational Modifications and Pathogenesis of Bacterial Meningitis Is a one shot Approach for Complete Mapping Realistic? [O37] Pascal Seyer - Identification and characterization of new protein partners of serotonin transporter [O29] Cellier Benoit XII (2) Manufacturer conference : Thermo
4:30pm
5:30pm
5:50pm 6:10pm
3:35pm-6:30pm 3:35pm
Cellier Benoit XII (2) 4:10pm
4:30pm
5:50pm
6:10pm 6:45pm-8:15pm 6:45pm-8:15pm
Chbr du Trsorier (7) Manufacturer conference : Waters
23/381
Wednesday September 21st

8:30am-9:25am Conclave (5) Plenary conference 3 - Guy BOUCHOUX - From the mobile proton
to wandering hydride ion: some mechanistic aspects of gas-phase ion chemistry [I3]
9:35am-12:30am 09:35am Conclave (5)
Session 5 - FT-ICR, Complexes Christian ROLANDO - Two dimensional FT-ICR, the only way to obtain
all the MS/MS correlations from a sample without selecting the parent ions. Instrumental developments and analytical applications [I17]
10:10am
Carlos Afonso - Identification par trs haute rsolution FT-ICR de

processus induits par la charge dans la dcomposition de peptides cycliques en ECD [O33]
10:30am
Break : Poster session n1 Coffee & Exposition Edith Nicol - Mechanisms leading to unusual ECD fragments: the ion
spectroscopy perspective [O11]
11:30am 11:50am 12:10am
Conclave (5)
Sbastien Schramm - Tabagisme actif/tabagisme passif, une tude

diffrentielle de la fraction particulaire [O15]
Elisabetta Boeri Erba - Quantifying Protein-Protein Interactions

Within Noncovalent Complexes Using Electrospray Ionization Mass Spectrometry [O3]
9:35am-12:30am 09:35am 10:10am Cellier Benoit XII (2)
Session 6 - Proteomics & Health Bruno DOMON - A Novel Strategy in Quantitative Proteomics: Its
Implication for Biomedical Research [I9]
Christophe D. Masselon - Bladder Cancer Biomarker Candidates

Discovery in a Multi-site Patients Cohort: a Label-free Quantitative Proteomics Study [O38]
10:30am Paneterie Grde Audience (12) 11:30am 11:50am 12:10am
Break : Poster session n1 Coffee & Exposition Willy V. Bienvenut - Proteomic targeting of angiogenic cell lines by
fumagillin mostly relies on the expression levels of METAP1 [O16]
Cellier Benoit XII (2)
Yannick Charretier -Microorganism characterization for the clinics

using SRM [O19]
Jrme Chenau - Dtection sensible et spcifique des spores de

Bacillus anthracis dans des matrices environnementales immunocapture et spectromtrie de masse en mode MRM [O28] par
Poster session n1 taking down / Poster session n2 hanging up 12:40am-1:55pm 12:55am-2:25pm 12:55am-1:50pm 1:55pm-2:25pm 2:00pm-2:25pm Jeanne Laurent (13) Cellier Benoit XII (2) Lunch Manufacturer conference : Bruker
Chbr du Trsorier (7) Manufacturer conference : Advion Chbr du Trsorier (7) Manufacturer conference : Promega Grde Audience (12) Exposition
24/381
Wednesday September 21st

2:30pm-3:25pm 3:35pm-6:30pm 3:35pm 4:10pm 4:30pm Paneterie (3) Grde Audience (12) 5:30pm Conclave (5) Conclave (5) Plenary conference 4 - Seth Grant - The organization of synapse proteomes [I11] Session 7 - Signaling Sacha BAGINSKY - Functional Proteomics: A Cornerstone in Plant Systems Biology [I1] Sonia Hem - Targeted proteomics profiling of the plasma membrane transportome in Arabidopsis thaliana [O20] Break : Poster session n2 Coffee & Exposition Franck Vandermoere - A quantitative phosphoproteomic approach
reveals differential phosphorylation of serotonin 2a receptor upon activation by hallucinogenic versus non-hallucinogenic agonists [O1]
5:50pm Conclave (5) 6:10pm
Jordane Biarc - The Induction of Serine/Threonine Protein Phosphorylations by Native and Mutant PDGFR/TrkA Chimeras in Stably Transfected PC12 Cells [O2] Sonia Cantel - Reflectron Positive ion Mode Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Sulfo-peptides [O8] Session 8 - Fragmentation Yury TSYBIN - Traveling to the Ends of the Proteome World. Positional N-terminal and C-terminal proteomics deciphers protein terminal and proteolytic post-translational modifications in the HPP [I18] Emmanuelle Sachon - Effets de lactylation des peptides de la peau de la rainette Pachymedusa dacnicolor pour un squenage de Novo par MALDI-TOF/TOF [O21]
3:35pm-6:30pm 3:35pm
Cellier Benoit XII (2) 4:10pm
4:30pm
Break : Poster session n2 Coffee & Exposition Corinne Bur - Structure Determination of Complex Plant Glycosphingolipids by Tandem Mass Spectrometry [O4]
5:30pm 5:50pm Cellier Benoit XII (2) 6:10pm
Stphane Bouchonnet - Investigating the unusual behavior of Metolachlor under chemical ionization in hybrid ion trap mass spectrometry [O12] Thierry Fouquet - Analytical strategy combining NMR, chemical
synthesis and mass spectrometry for molecular and structural characterization of plasma-polymerized siloxanes [O23]
6:30pm-7:55pm 6:30pm-7:55pm 8:00pm-11:30pm
Cellier Benoit XII (2)
SFSM Meeting
Chbr du Trsorier (7) SFEAP Meeting Jeanne Laurent (13) Gala dinner
25/381
Thursday September 22nd

8:30am-9:25am 9:35am-12:15am 09:35am 10:10am Conclave (5) Conclave (5) Plenary conference 5 - Olga VITEK - Statistical methods and tools for protein quantification in MS-based proteomics [I19] Session 9 - Bioinformatics Oliver KOHLBACHER - Automated high-throughput analysis of quantitative proteomics data [I14] Christine Carapito - Une suite logicielle pour la protomique interface sur une grille de calcul. Utilisation d'algorithmes libres pour l'identification MS/MS, le squenage de novo et l'annotation fonctionnelle [O30] Break : Poster session n2 Coffee & Exposition Markus Muller - Evaluating the accuracy of the positioning of phosphorylations by MS/MS spectra [O24] Conclave (5) Benoit Valot - Nouveaux algorithmes de regroupement pour grer l'identification des protines ou de modifications posttraductionnelles dans les analyses de protomique haut dbit [O36] Session 10 - Metabolomics Pascal KINTZ - Mass spectrometry in forensic hair testing : example of drug-facilitated crimes [I13] Emilien Jamin - Dveloppement dune mthode mtabolomique associant la RMN et lUHPLC-HRMS [O9] Break : Poster session n1 Coffee & Exposition Sylvain Chreau - Prises dempreintes urinaires par LC-HRMS : La mtabolomique, une stratgie innovante pour le dpistage de lutilisation danabolisants en levage [O10] Agnta Kiss - Lapport de la spectromtrie de masse haute-rsolution
lobtention dempreintes molculaires d'chantillons durine. Applications potentielles au dopage [O35]
10:30am Paneterie (3) Grde Audience (12) 11:30am 11:50am
9:35am-12:15am 09:35am 10:10am 10:30am Paneterie (3) Grde Audience (12) 11:30am Cellier Benoit XII (2) Cellier Benoit XII (2)
de
11:50am
12:15am-1:30pm 1:30pm-2:15pm
Jeanne Laurent (13)
Lunch Plenary conference 6 - Virginie BRUN PSAQ standards for accurate quantification of proteins: from the concept to clinical application [I5]
2:15pm-2:30pm 2:30pm-3:25pm
Conclave (5)
SFSM / SFEAP prizes Closing conference - Pierre CHAURAND MALDI Imaging Mass Spectrometry: Principle, State of the Art and Future Challenges [I6] Closing
3:30pm
26/381
Invited conference
27/381
[I1] Functional Proteomics: A Cornerstone in Plant Systems Biology

Sacha BAGINSKY Martin-Luther-Universitt Halle-Wittenberg, Institut fr Biochemie und Biotechnologie, Abteilung Pflanzenbiochemie, Weinbergweg 22 (Biozentrum), 06120 Halle (Saale), Deutschland Contact : sacha.baginsky@biochemtech.uni-halle.de
Different functional proteomics tools are now available that enable the quantitative characterization of proteome dynamics and the mapping of posttranslational modifications. We report here examples how we used these tools to characterize the functional proteome of Arabidopsis and rice cell organelles, with a focus on plant-specific plastids. We analyzed the Arabidopsis proteome at genome-scale and provide quantitative information about organellar proteomes in different plant organs by normalized spectral counting (Baerenfaller et al., Science 320, 938-41; Baerenfaller et al., Integrative Biology 3, 225-237). For a functional characterization of plastid protein import, we analyzed the proteomes of protein import mutants and established protein N-termini to distinguish precursor from mature plastid proteins in WT, ppi1 and ppi2. These analyses revealed the accumulation of precursor proteins in the cytosol of protein import mutants. In order to assess the short-term regulation of the chloroplast proteome in response to environmental signals, we analyzed the chloroplast phosphoproteome and characterized its dynamics during a circadian cycle (Reiland et al., Plant Physiol. 150, 889-903). Phosphorylation motif utilization suggests that the phosphorylation network topology in chloroplasts is characterized by many-to-many relationships. To establish the kinase/substrate nodes in this network we performed comparative quantitative phosphoproteome profiling experiments with wildtype and kinase mutant plant material, starting out with the STN8 kinase. Differential protein phosphorylation was assessed by relative quantification with extracted ion chromatograms and the data were further validated by functional characterization of selected substrates (Reiland et al., Proc. Natl. Acad. Sci. USA, in press). We present here our data, comment on reliability and reproducibility and propose strategies to increase both at a reasonable cost.
28/381
[I2] Ion Mobility Mass Spectrometry for Intrinsically Disordered Proteins a SWOT analysis
Perdita BARRAN The EastChem School of Chemistry, The University of Edinburgh, Edinburgh, UK Contact : perdita.barran@ed.ac.uk
The p53 protein is a transcription factor which plays a central role to tumour suppression. Loss of p53 function is correlated with the development of cancer in which the MDM2 protein binds to the N-terminal transcription activation domain shutting down function 3. Mass spectrometry studies of WT p53 and mutants of the DNA binding domain following nanoelectrospray from native conditions show a wide charge state range for charge states n= 8 - 18 for monomeric species of the general form [M+nH] n+. Mass spectrometry data from both instruments are qualitatively similar. The most abundant peaks are at n =9 and n=10 indicating the dominance of a compact structure. Multimeric species are observed for the WT and mutants, however the dimers shown in the WT are more intense. Generally, the collision crosssections of the monomer are observed to increase with increasing charge in a stochastic fashion attributed to protein unfolding due principally to coulombic repulsion, which can be attributed to the plasticity of these IDPs . Multiple gas-phase conformers are resolved for a number of charge states, over a very wide charge state range. Subtle differences in unfolding transitions between the WT and mutated samples can be characterized. For instance the H115N mutant, altered at the boundary of loop I seems to be less structured favoring the more extended conformations when compared with WT p53.,Conversely, the R249A/H115N mutant appears to be more compact. The thermally induced unfolding also shows interesting trends, and in particular reveals the resistance to unfolding of this important IDP. These results are interpreted in terms of the biological activity of these proteins and also in terms of the implications for the use of IM-MS to study this largely ignored, but critically important, class of proteins.
29/381
[I3] From the mobile proton to wandering hydride ion: some mechanistic aspects of gas-phase ion chemistry
Guy BOUCHOUX Ecole Polytechnique, Laboratoire des Mcanismes Ractionnels (DCMR), Dpartement de Chimie, 91120 Palaiseau, France; CNRS, UMR 7651, Palaiseau Cedex, France Contact : bouchoux@dcmr.polytechnique.fr
Structural characterization of molecular species by mass spectrometry supposes the knowledge of the type of ions generated and the mechanism by which they dissociate. In this context, a need for a rationalization of ESI(+)(-) mass spectra of small molecules has been recently expressed [1]. Similarly, at the other end of the mass scale, efforts are currently made to interpret the major fragmentation processes of protonated and deprotonated peptides and their reduced forms produced in electron capture (ECD) or electron transfer (ETD) experiments [2,3]. Most fragmentation processes of molecular and pseudo-molecular ions may be described by a combination of several key mechanistic steps: simple bond dissociation, hydrogen atom, hydride ion or proton migrations, formation of ion-neutral complex intermediates Selected crucial aspects of these elementary reactions, occurring inside positively charged ions, will be recalled and illustrated by examples taken in the recent mass spectrometry literature. Emphasis will be given on the protonation process and its consequences in term of structure and energetic [4]. 1 WMA. Niessen. Mass Spectrom Rev 2011, 30, 626-663. 2 AG Harrison. Mass Spectrom Rev 2009, 28, 640-654. 3 SA McLuckey, M Mentinova. J Am Soc Mass Spectrom 2011, 22, 3-12. 4 G Bouchoux. Mass Spectrom Rev 2007, 26, 775-835.
30/381
[I4] Development and Applications of Photodissociation for Biological Applications

Jennifer BRODBELT Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712 Contact : jbrodbelt@mail.utexas.edu
The tremendous growth in the application of mass spectrometry for detection, quantification and characterization of biological molecules has spurred the exploration of new ion activation/dissociation methods. Although collision induced dissociation (CID) remains the gold standard for structural characterization of ions, it has several shortcomings (e.g. insufficient energy deposition, limited applicability for pinpointing post-translational modifications in peptides, etc.) that have stimulated the search for other activation methods. Photodissociation offers an especially promising alternative to traditional CID in ion traps, in addition to its success with FTICR and time-of-flight instruments. There are several compelling advantages of using lasers to activate ions in a mass spectrometer. Photoactivation is a non-resonant process, meaning that both the selected precursor ions and primary fragment ions may be activated, leading to secondary dissociation and a richer array of fragment ions without requiring deliberate sequential stages of ion manipulation. The ability to vary energy deposition without resorting to multi-stage experiments (MS n) is particularly important when analyzing macromolecules in which initial ion currents may be low and the total sample quantity is limited. The reduction of ion losses is a critical feature when analyzing small ion populations while maintaining adequate detection sensitivity. Photoactivation can be utilized to analyze both negative and positive ions, so it offers the potential for broader characterization of the proteome. UV photodissociation using a laser offers fast, high energy deposition that yields good sequence coverage for peptide analysis.
31/381
[I5] PSAQ standards for accurate quantification of proteins: from the concept to clinical application
Virginie BRUN Laboratoire d'Etude de la Dynamique des Protomes, Unit de Biologie Grande Echelle, CEA/INSERM/UJF 1038, 17, rue des Martyrs, 38054 cedex 9 Grenoble, France Contact : virginie.brun@cea.fr
Technological developments that will provide reliable and multiplex quantification of proteins in biofluids are critical for biomarker research and medical science. Mass-spectrometry analysis is currently attracting considerable interest due to its multiplexing capacities. Combined with stable isotope-labelled quantification standards [1], MS-based assays can provide quantitative data for hundreds of peptides generated by trypsin digestion of proteins. However, to envision MS as a relevant methodology for biomarker evaluation and biomarker measurement in clinical laboratories, some analytical variabilities still need to be resolved [2]. In 2007, we developed the PSAQ TM method (Protein Standard Absolute Quantification) which uses full-length isotope-labelled protein standards to quantify target proteins [3]. In this presentation, well demonstrate the specific advantages of the PSAQ TM method for accurate and reliable quantification of protein biomarkers. Recent technological advances related to the production and use of PSAQ TM standards will be presented. Several applications of the PSAQ TM method in the health domain will also be exposed. References 1. Brun V. et al., (2009) Isotope dilution strategies for absolute quantitative proteomics. J. Proteomics 72, 740-749. 2. Hoofnagle A.N. et al., (2010)Quantitative Clinical Proteomics by Liquid ChromatographyTandem Mass Spectrometry: Assessing the Platform. Clin. Chem. 56, 161-164. 3. Brun V. et al (2007) Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol. Cell. Proteomics 6, 2139-2149.
32/381
[I6] MALDI Imaging Mass Spectrometry: Principle, State of the Art and Future Challenges
Pierre CHAURAND Dpartement de chimie, universit de Montral. Montral, Qubec, Canada Contact : pierre.chaurand@umontreal.ca
MALDI-based imaging mass spectrometry is a new technology that allows to map different biocompounds and xenobiotics directly from thin tissue sections. Numerous classes of biomolecules including metabolites, phospholipids, peptides and proteins can be detected and mapped in direct correlation with the underlying histology. Molecular profiles and images depend on the types of tissues or cells studied and certain signals can be directly correlated with the health status of the tissue specimen. Indeed, the technology is sufficiently sensitive to detect variations in the molecular composition induced by the presence of disease or by drug uptake. Numerous technical advances such as automated matrix deposition and the development of in situ chemistries now allow us to study the proteomic content of fresh frozen and formalin fixed paraffin embedded tissue specimens. After an introduction of the technology and a description of current progresses the different fields of research of imaging mass spectrometry will be presented. In particular its enormous potential in clinical settings in complement to traditional histopathology and its important role in the study of drug distribution and effects in various biological tissues will be described. Finally, a critical outlook will be made towards the developments to be made for the technology to become a mainstream analytical tool.
33/381
[I7] Ambient Ionization and Miniature Mass Spectrometers

Graham COOKS Department of Chemistry, Purdue University, West Lafayette IN 47907, US Contact : cooks@purdue.edu
The recent development of ambient ionization methods is transforming the applications of mass spectrometry (MS) allowing virtually any sample to be examined in air, rather than being introduced into the vacuum system. This talk focuses on two ambient ionization methods: (i) paper spray (PS) in which in which samples are ionized in air, directly from filter paper (and similar materials including whole plant or animal tissue) and (ii) desorption electrospray ionization (DESI) in which the sample is impacted by charged microdroplets which pick up analyte by dissolution and carry it to the MS. The physical basis of each of these experiments is described. DESI finds application in disease diagnosis by tissue imaging and examples of human bladder, liver and brain cancer diagnostics will be given. DESI imaging combines the chemical information collected for multiple analytes from the mass spectrometer with spatial information, which makes it useful for analyzing histological sections of biological tissue. Alterations in the distribution of polar lipids are associated with malignant transformations in tissue. Multivariate statistical analysis using principal component analysis (PCA) is used to analyze the imaging MS data, magnifying differences between the lipid profiles of tissue as a function of disease state. Data showing glioma diagnosis and prostate biomarker recognition will be emphasized. Whole blood analysis for therapeutic agents is achieved in a few seconds using paper spray. Remarkably, the method is quantitatively accurate and precise and covers the therapeutic range for many oncology drugs. The promise of this method for point-of-care diagnostics will be shown. LTP applications in food safety and bacterial identification also will be described. Ultimately ambient ionization methods are best used with handheld miniature mass spectrometers, a combination that has enormous potential to transform in situ chemical analysis. Progress in interfacing ambient ionization sources to miniature mass spectrometers is described. The support of this work by NSF, DOE and the Alfred Mann Foundation at Purdue is gratefully acknowledged as is ongoing collaboration with Prof. Zheng Ouyang.
34/381
[I8] Proteomics analyzes reveal unexpected aspects of the evolution of phagosomes over 1.2 billion years
Jonathan Boulais, Matthias Trost, Pierre Thibault, Michel Desjardins Dpartement de Pathologie et Biologie Cellulaire, Universit de Montral, Montral, Qubec, Canada Contact : michel.desjardins@umontreal.ca
Phagosomes are intracellular organelles formed in a variety of cells following the engulfment of large particulate materials by phagocytosis. This organelle plays key roles in both innate and adaptive immunity by promoting the establishment of the molecular properties needed to kill microorganisms and present some of their antigens on MHC molecules. In the last 20 years, the striking advances in the field of proteomics and systems biology have largely contributed to our understanding of the molecular mechanisms regulating phagosome functions. This presentation will highlight how the application of large-scale approaches has contributed to reveal novel aspects of the functional properties of phagosomes and their emergence during evolution.
35/381
[I9] A Novel Strategy in Quantitative Proteomics: Its Implication for Biomedical Research
Bruno DOMON Luxembourg Clinical Proteomics Center, CRP-Sant, Strassen, Luxembourg Contact : bdomon@crp-sante.lu
The generation of accurate and comprehensive data sets has become an essential element of systems biology and biomarker studies. While large scale discovery experiments one hand and targeted quantitative analyses on the other are nowadays widely used in proteomics, we are proposing an alternative approach, which is based on a novel quadrupole/orbitrap instrument. The high acquisition speed and the exquisite sensitivity of such a hybrid mass spectrometer allow performing reliable qualitative and quantitative experiments. The additional selectivity intrinsic to the high resolution orbitrap mass analyzer enables the development of novel quantification methods. Quantitative experiments can be performed either in full scan mode (using the high-resolution / accurate mass capability) or in MS/MS mode by analyzing specific fragment ions (i.e. SRM-like mode). The different modes of operation, their advantages and limitations will be presented in details. This technique has been applied to precisely quantify biomarker candidates in bodily fluids, and more specifically in urine samples. The quantitative analyses were performed in conjunction with stable isotope dilution, using second generation synthetic polypeptides, composed of one universal reporter fragment facilitating the systematic, precise quantification of multiple analytes in complex biological samples.
36/381
[I10] A systematic screen for proteinlipid interactions in Saccharomyces cerevisiae

Anne-Claude GAVIN Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany Contact : gavin@embl.de
Biological function emerges from the concerted action of numerous interacting biomolecules. Deciphering the molecular mechanisms behind cellular processes requires the systematic charting of the multitude of interactions between all cellular components. While proteinprotein and protein DNA networks have been the subject of many systematic surveys, others critically important cellular components, such as lipids, have to date rarely been studied in large-scale interaction screens. The importance of proteinlipid interactions is evident from the variety of protein domains that have evolved to bind particular lipids and from the large list of disorders, such as cancer and bipolar disorder, arising from altered proteinlipid interactions. The importance of lipids in biological processes and their under-representation in current biological networks suggest the need for systematic, unbiased biochemical screens. Here, we report a screen to catalog protein lipid interactions in yeast using a lipid arrays. To illustrate the data sets biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. The dataset presented here represents an excellent resource to enhance the understanding of lipids function in eukaryotic systems.
37/381
[I11] The organization of synapse proteomes

Seth Grant Wellcome Trust Sanger Institute, Cambridge, Edinburgh University, Edinburgh Contact : sg3@sanger.ac.uk
Proteomic mass spectrometry has been a groundbreaking technology for studying the composition of complex subcellular organelles. In the nervous system, proteomics has discovered more synaptic proteins than any other method and uncovered a remarkable complexity in the signaling machinery involved in the communication between nerve cells. These data sets have opened up new insights into the evolution of the brain, the organisation and architecture of complex circuits and in the diversity of behavior. The presentation will show how synapse proteomics has introduced molecular complexity into neuroscience and how from this complexity simple organizational features and novel mechanisms have emerged. Synapse proteomics has allowed a unique convergence with human genetics leading to the identification of subsets of proteins that control phenotypes that are involved with many brain diseases. The study of the human synapse proteome has revealed that postsynaptic proteome is disrupted by over 200 mutations involved with over 130 brain diseases.
38/381
[I12] Charge Detection Mass Spectrometry

Martin JARROLD Chemistry Department, Indiana University, Bloomington, Indiana 47401, USA Contact : mfj@indiana.edu
This presentation will provide an overview of recent work pushing the boundaries of sensitivity and accuracy with charge detection mass spectrometry. In conventional mass spectrometry the m/z distribution is obtained for an ensemble of ions, z is then deduced from the m/z distribution of the ensemble, and the ion mass is obtained from the corresponding m/z and z. This approach only works when the charge states are resolved in the m/z distribution, which is usually not the case for large and heterogeneous ions. In charge detection mass spectrometry, m/z and z are directly measured for each individual ion, so that the mass can be determined for each ion. This approach allows true mass spectra to be determined for very large and very heterogeneous ions. The two main challenges with charge detection mass spectrometry are the sensitivity and accuracy of the charge measurements. Efforts to attain an accuracy of a single elementary charge (1e) and a sensitivity of 10e will be described. Applications to ions with masses from kilodaltons to teradaltons will be demonstrated.
39/381
[I13] Mass spectrometry in forensic hair testing : example of drug-facilitated crimes

Pascal KINTZ X-Pertise Consulting, 84 route de Saverne, 67205 Oberhausbergen, France Contact : pascal.kintz@wanadoo.fr
The use of a drug to modify a persons behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drugfacilitated crimes (sexual assault, robbery) has caused alarm in the general public. Drugs involved can be pharmaceuticals, such as benzodiazepines (flunitrazepam, lorazepam ...), hypnotics (zopiclone, zolpidem), sedatives (scopolamine, neuroleptics, some anti-H1) or anaesthetics (GHB, ketamine), drugs of abuse, such as cannabis, ecstasy or LSD, or more often ethanol. To perform successful toxicological examinations, the analyst must follow some important rules : 1. obtain as soon as possible the corresponding biological specimens (blood, urine and hair), 2. use sophisticated analytical techniques (LC/MS, HS/GC/MS, tandem mass spectrometry); and 3. take care on the interpretation of the findings. Even after the publication of these guidelines for the clinicians, in most cases specimens are collected at best 24 hours after the crime has occurred. Drugs used to facilitate sexual assaults can be difficult to detect (active products at low dosages, chemical instability), possess amnesic properties and can be rapidly cleared from the body (short half-life). Prohibiting immunoassays and using only hyphenated techniques, substances can be found in blood for 6 hours to 2 days and in urine for 12 hours to 5 days. In these situations, blood or even urine can be of poor interest. This is the reason why this laboratory developed an original approach based on hair testing. Hair was suggested as a valuable specimen in situations where, as a result of a delay in reporting the crime, natural processes have eliminated the drug from typical biological specimens. While there is a lot of papers focused on the identification of drugs in hair following chronic drug use, those dealing with a single dose are very scarce. This laboratory recommends to wait for 3-4 weeks after the offense and then collect 4 strands of about 100 hair. One strand will be used to test for drugs of abuse (mostly for cannabis, but also for ecstasy related compounds and cocaine that are sometimes observed), one for GHB and the other one for a screening of 30 various sedatives. The last strand can be used for a potential counter-analysis. After decontamination, hair is then segmented as follows : 0 to 2 cm (segment corresponding to the period of crime), 2 to 4 and 4 to 6 cm (which should be drug-free). For GHB, segments are of 3 mm (n=8). Conventional GC/MS can be used to test for drugs of abuse, but given the expected concentrations to measure in low weight segments (in order to avoid the shave the victim), GHB and sedatives are tested by GC-MS/MS and UPLC-MS/MS, respectively. The experience of the authors will be documented in cases involving GHB, zolpidem, bromazepam, alprazolam, scopolamine, alimemazine, diphenhydramine ... Hair analysis may be a useful adjunct to conventional drug testing in sexual assault. It should not be considered as an alternative to blood and urine analyses, but as a complement. This approach may find useful applications, but appears very expensive, given the number of analyses to achieve with sophisticated equipment.
40/381
[I14] Automated high-throughput analysis of quantitative proteomics data

Oliver KOHLBACHER Applied Bioinformatics, Center for Bioinformatics, Department of Computer Science, University of Tbingen, Sand 14, 72076 Tbingen, Germany Contact : oliver.kohlbacher@uni-tuebingen.de
Over the last decade HPLC-MS has become the workhorse in proteomics and metabolomics. Increasing sensitivity and resolution of the instruments give us unprecedented coverage of the proteomes and metabolomes under investigation. The flip side of this development is the amount and complexity of data produced. Similar to what we observe in next-generation sequencing, bioinformatics analysis of a high-throughput experiment is rapidly becoming the bottleneck. In this talk, I will illustrate how wet-lab workflows have to be matched by appropriate data analysis workflows in order to enable more complex experiments. Based on our software package OpenMS/TOPP, I will illustrate how analysis workflows can be set up, adapted to specific experimental designs for different quantification strategies. The resulting analyses can then be run automatically. Automated workflows also enable more compute-intensive methods, as computer power scales much better than manual labor done by PhD students and postdocs. I will give a few examples, how these more complex analyses can result in a drastic increase in the coverage and accuracy of the experiment.
41/381
[I15] Traveling to the Ends of the Proteome World. Positional N-terminal and Cterminal proteomics deciphers protein terminal and proteolytic posttranslational modifications in the HPP
Christopher OVERALL UBC Centre for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, B.C. V6T 1Z3 Canada Contact : chris.overall@ubc.ca Protein termini are truncated by proteolysis, but the extent to which this molds the proteome in vivo is unknown. In addition to constitutive proteolysis during protein synthesis and maturation, the processing of a mature protein often irreversibly changes its activity. Specific degradomics techniques are needed to rapidly identify and quantify the N- and C-terminomes in order to reveal the extent of post-translational modifications of protein termini and therefore the functional state of key molecules, the extent of proteolysis in a system, and to identify new protease substrates. We have devised new approaches to enrich for the N and C termini of proteins for high throughput terminome analyses of human tissues and cells for the Human Proteome project, in particular for chromosome 21 that is Canadas focus for the HPP. Broad coverage Nterminome analysis necessitates a negative selection procedure as the variety of original mature protein Nterminal blocked peptides each present individual chemical hurdles for their enrichment by positive selection strategies. We developed a combined N-terminomics and C-terminomics and protease substrate discovery degradomics platforms for the simultaneous quantitative analysis of the N-terminome and proteolysis on a proteome-wide scale called Terminal Amino Isotopic Labelling of Substrates (TAILS, Kleifeld et al Nature Biotech 28, 281-288; Prudova et al 2010 Mol Cell Proteomics; auf dem Keller et al 2010 Mol Cell Proteomics) and C-TAILS (Schilling et al Nature Methods 2010). By using novel polymers to deplete the internal tryptic peptides, TAILS suffers little from sample loss and low yields, so requiring only 100 microgram of sample and one MS/MS analysis per sample. By a three-day procedure with flexible labelling options, TAILS can be adapted to a variety of experimental situations including cell culture and complex biological sample analysis. Incorporating iTRAQ labelling iTRAQ-TAILS also provides wide coverage of all forms of naturally blocked N-terminal peptides and allows for their quantification through labelling of lysine side-chains in up to 8 samples. In addition to providing valuable proteome annotation this has several unique advantages. TAILS permits exploitation of the acetylated and other blocked mature protein N-terminal peptides as a statistical classifier that is then used to set isotope ratio cut offs that reveal protease activity. We introduce a novel parameter evaluating ion intensity dependent quantification confidences of single peptide quantifications. Being a quantitative procedure, TAILS can analyse the substrate degradome of a broad specificity protease or one with no known specificity without manual data parsing, in the same experiment, and also do this in vivo. We have applied TAILS to a variety of proteases and compared protease knock out mice in models of arthritis, skin inflammation and models of breast cancer metastasis and pancreatic carcinoma in the RIP-Tag model. Typical analyses identify over 3000 N-terminal peptides from which we found that the removal of the N-terminal methionine is dependent upon the amino acid at position 2 with distinct preferences found for valine, glycine, alanine and serine. In one experiment, acetylation occurred on 731 original mature protein N-terminal peptides but at the initiator methionine in only 153 of these instances. In 578 cases, acetylation was at position 2 in the protein after removal of 1Met, with alanine, serine and methionine being the preferred acetylated residues. Internal acetylation sites exhibit a distinct acetylation pattern that differs from the N-terminal acetylation. Finally N-terminal positional proteomics enables MS sample simplification with proteins identified in bronchoalvelar fluid and serum having abundances spanning a range greater than six orders of magnitude. This underscores the potential of TAILS to tackle the dynamic range analysis problem in complex proteomes and as a high throughput approach annotate the N and C termini in the HPP. Link : http://www.clip.ubc.ca
42/381
[I16] Targeted proteomics from proteins to proteome maps : potential and bottlenecks
Paola PICOTTI Institute of Biochemistry, Department of Biology, ETH Zuerich, Zuerich. Switzerland Contact : paola.picotti@bc.biol.ethz.ch
Selected Reaction Monitoring (SRM) is a targeted mass spectrometry technique which emerged in the field of proteomics as a complement to the untargeted shotgun methods. The main advantages of SRM become apparent when predetermined sets of proteins need to be measured across multiple samples in a consistent and accurate manner. The technology is however still in its infancy for protein analysis and several challenges need to be addressed to demonstrate the power of the technique in biological research and increase its widespread application in non-specialized laboratories. To evaluate the applicability of SRM to studying biological networks, we applied it to the analysis of a metabolic network constituted by proteins covering a broad range of abundances and including a large number of families of isoenzymes, sharing high sequence overlap. Proteins in the network were quantified by SRM in yeast cells grown under a series of conditions inducing radically different metabolic setups and in a growth time-course of cells transiting through a series of metabolic phases. The quantitative dataset generated highlighted how yeast metabolism adapts to changing conditions of supply and demand of nutrients and suggested differential functionality for several isoenzymes. The application showed the potential of the technique to elucidate the dynamics of cellular networks through large number of perturbing conditions. In order to expand the capabilities of the technique, we used an approach based on libraries of unpurified synthetic peptides to develop at high-throughput SRM assays for entire proteomes. We used the method to develop SRM assays for the ~6,000 proteins that constitute the proteome of S. cerevisiae and then expanded it to the generation of SRM assays for >90% of the human proteome. The synthetic peptide libraries were also used to generate gold-standard ion trap spectral libraries to be used for spectral matching of shotgun proteomic datasets in discovery-driven experiments. The described SRM assays and spectral libraries are currently being made publicly available via the SRMAtlas user interface, which supports their dissemination across different laboratoreis. The potential of such proteome maps, for targeted and discovery proteomics, as well as their limitations will be discussed.
43/381
[I17] Two dimensional FT-ICR, the only way to obtain all the MS/MS correlations from a sample without selecting the parent ions. Instrumental developments and analytical applications
Christian ROLANDO Universit Lille 1 Sciences et Technologies, USR CNRS 3290 Miniaturisation pour l'Analyse, la Synthse & la Protomique (MSAP), 59655 Villeneuve d'Ascq Cedex, France Contact : Christian.Rolando@univ-lille1.fr
The pulse sequence for 2D FT-ICR MS was first developed in 1988 [1] by MS and NMR teams respectively lead by Tino Gumann and Geoffrey Bodenhausen [1] based on a key experiment demonstrating cyclotron excitation reversibility [2] and conveys detailed information for compounds in complex samples in one easily readable 2D mass spectrum without ion isolation. We revisited 2D FT-ICR MS [3] which, unlike 2D NMR, has no analytical applications yet. Gas-free fragmentation modes allow us to retain high resolution and sensitivity, and the advances in electronics and computer technology since 1988 enable broadband acquisition. The method, however, needs to be optimized in terms of scintillation noise, experiment time and necessary amounts of sample. In this study, we apply techniques adapted from NMR spectroscopy to optimize 2D FT-ICR MS. Because in both dimensions the spectrum is obtained after Fourier transforming a signal that has been sampled at regular time intervals (in the vertical dimension, a delay, and in the horizontal dimension, a measurement date), the resolution of 2D mass spectra behaves in both dimensions like the resolution in onedimensional FTMS: it increases with the number of data points and is inversely proportional to m/z ratio. In preliminary experiments, time transients were limited to 32k data points since the 32-bit-written software cannot handle files larger than 256 Mb. Using 2048 scans leads to hour-long experiments with a resolution of ~1000 in the MS and fragment ion MS/MS dimension but only a few hundred in the correlation parent ion dimension [3]. For example, we were able to distinguish the fragments of the 79Br and 81Br isotopes and the 12 C and 13C isotopes of singly charged ions of bromopride, a small brominated drug (molecular weight 344.25 g.mol-1). The new 64-bit version of the NPK package developed by Marc-Andr Delsuc [4] and NMRTEC allows us to work on files larger than 16 Gb. An experiment on intact cytochrome C (molecular weight 12 kDalton) using 8192 scans of 512k points time transients shows line base separation for each multicharged ion bearing around 10 charges (ca 10 000 resolution) and quadrupole like (1 m/z unit) separation in the correlation dimension (ca 1000 resolution). Furthermore these experiments show that the resolution in both dimensions remains inversely proportional to m/z ratio, as expected. All of our experiments were performed using nanoESI at concentrations of 1 pmol/L, leading to sample consumption of less than 20 pmol per hour per experiment, but at the expense of signal intensity fluctuation which translates into scintillation noise in the 2D data. We adapted the NMR version of the Cadzow denoising algorithm [5] for 2D FT-ICR mass spectra. This algorithm is based on the decomposition of the signal in singular values from which the signal at longer time may be predicted. The Cadzow noise reduction dramatically reduced fluctuations and brought out additional fragmentation peaks that were masked by the noise [6]. Results obtained using IRMPD and ECD fragmentation without any in-cell or quadrupole separation for simple compounds like individual peptides, the multicharged ion distribution of small proteins and more complex mixtures like tryptic protein digests will be presented. Finally future development of 2D FT-ICR will be briefly discussed at the sight of multidimensional NMR methodologies developed during the last twenty years. [1] P. Pfndler et al., J. Am. Chem. Soc. 110 (1988) 5625-5628 [2] A.G. Marshall et al., Chem. Phys. Letts. 105 (1984) 233-236 [3] M.A. van Agthoven et al., Int. J. Mass Spectrom., doi:10.1016/j.ijms.2010.10.034 [4] D. Tramesel et al., J. Magn. Res. 188 (2007) 56-67 [5] C. Brissac et al., J. Biomol. NMR 6 (1995) 361-365 [6] M.A. van Agthoven et al., Rapid Commun. Mass Spectrom., 25 (2011) 16091616.
44/381
[I18] High resolution mass spectrometry at high speed: advances in methods, techniques and applications
Yury TSYBIN Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fdrale de Lausanne, CH-1015, Lausanne, Switzerland Contact : yury.tsybin@epfl.ch
High magnetic field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides the unbeatable analytical performance in terms of high resolution measurements. However, the high resolution measurements require a long experimental time, e.g., 1-20 seconds per single mass spectrum acquisition, and thus are incompatible with the current and near-future requirements for the high throughput and fast data acquisition, enforced by the short peptide and protein elution time from the chromatographic column. Here, we will discuss some of the possible solutions to this problem from both, hardware and signal processing development. The FT-ICR MS hardware development follows the implementation of ultra-high, e.g., 21 T, magnetic field environments together with higher acquisition speed and harmonized ICR ion traps. The recent progress in Orbitrap FTMS development has significantly reduced the gap between Orbitrap and ICR FTMS in terms of obtained resolving power required for the mainstream MS applications, 60-200k. Finally, the state-of-the-art time-of-flight (TOF) mass analyzers have demonstrated a substantial progress in the recent years and now offer 30-60k resolving powers achieved at comparable times to the high field ICR and Orbitrap FTMS and faster. On the other hand, incredible progress in the computational power and high frequency electronics opens the doors to the advanced signal processing development, which received a particular attention in the recent years. A number of groups, including ours, are in the process of tailoring the super-resolution signal processing methods, e.g., filter diagonalization method (FDM), to the needs of the ICR and Orbitrap MS. The goal is to replace the FT-based signal processing with the methods that would require 10-100 times shorter transient time-domain signals to yield a similar level of the resolving power.
45/381
[I19] Statistical methods and tools for protein quantification in MS-based proteomics
Olga VITEK Department of Statistics & Department of Computer Science, Purdue University, West Lafayette, IN 47907 Contact : ovitek@purdue.edu
The goal of many proteomic experiments is to quantify and compare the abundances of proteins in complex biological mixtures. This can be accomplished in a variety of mass spectrometry-based workflows, such as label-free or label-based LC-MS workflows, or label-free or label-based SRM workflows. Although the experimental details of these workflows vary greatly, they all output a list of identified and quantified spectral features. There is currently no consensus on how to appropriately handle the repeated quantitative measurements on a protein in a sample, and how to derive protein-level conclusions across all labels, features, samples and conditions. We propose a general statistical modeling framework for protein quantification based on linear mixed-effects models. It is applicable to most experimental designs, LC-MS and SRM experiments, and label-based and label-free SRM workflows. We illustrate the utility of the framework in a series of investigations with fairly complex designs: a 3-way factorial label-free LC-MS, and a label-based SRM time course investigation of central carbon metabolism of S.cerevisiae. We further illustrate the sensitivity and specificity of the framework using controlled spike-in experiments, and using a series of simulated datasets. Finally, we discuss the freely available software that we developed to implement the modeling framework.
46/381
Selected short oral communications
47/381
[O1] A quantitative phosphoproteomic approach reveals differential phosphorylation of serotonin 2a receptor upon activation by hallucinogenic versus non-hallucinogenic agonists
Franck Vandermoere1, Samah Karaki1, Clotilde Mannoury la Cour2, Carine Becamel1, Joel Bockaert1, Mark J Millan2, Philippe Marin1
1 2
Institut de Gnomique Fonctionnelle, CNRS UMR5203, 141 rue de la Cardonille, 34094 Montpellier, France IDR Servier, 125 chemin de Ronde, 78290 Croissy/Seine, Paris, France
Contact: Franck Vandermoere (franck.vandermoere@igf.cnrs.fr)
Keywords: Signaling, interactomics and post-translational modifications The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamide (LSD), which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity do not. Using quantitative phosphoproteomics combining SILAC, phosphopeptide enrichment by hydrophilic interaction chromatography /immobilized metal affinity chromatography and FT mass spectrometry, we compared the phosphoproteome in cells transiently expressing the 5HT2A receptor under three conditions: no-stimulation; exposure to the hallucinogen DOI and exposure to the non-hallucinogenic agonist lisuride. Among the 5,996 identified phosphopeptides, 454 sites were differentially phosphorylated upon exposure to DOI vs. lisuride. These include a serine phosphorylated upon exposure to DOI but not to lisuride and located in the i3 loop, a region important for 5HT2A desensitization. Mass spectrometry analysis of immunopurified receptor further confirmed such a differential phosphorylation. Correspondingly, exposure to hallucinogens induced a less pronounced receptor desensitization than exposure to non-hallucinogenic agonists. In conclusion, this phosphoproteomic analysis reveals that 5-HT2A receptor stimulation by hallucinogenic vs. non hallucinogenic agonists induces contrasting phosphorylation patterns that may be related to their distinct behavioural responses. It also provides the first demonstration of differential phosphorylation of a G protein-coupled receptor upon stimulation by biased agonists. Link: http://www.igf.cnrs.fr/spip.php?article89
48/381
[O2] The Induction of Serine/Threonine Protein Phosphorylations by Native and Mutant PDGFR/TrkA Chimeras in Stably Transfected PC12 Cells
Jordane Biarc1, Robert Chalkley1, Al Bulingame1, Ralph Bradshaw1
1
Mass spectrometry facility-UCSF, 600th street, 94158-2517 San Francisco, USA
Contact: Jordane Biarc (jordane.biarc@hotmail.fr)
Keywords: Signaling, interactomics and post-translational modifications The tyrosine kinase receptor (RTK) TrkA, which is activated by NGF (nerve growth factor) binding, plays a major role in differentiation and survival of both peripheral and central nervous system neurons. When PC12 cells, a model paradigm for these functions, are stimulated with NGF, they extend neurites and acquire characteristics similar to sympathetic neurons. After stimulation, TrkA undergoes tyrosine autophosphorylation creating docking sites that link several effectors and adaptors to the activation of downstream signaling pathways, which are characterized by a second, longer lasting, wave of protein phosphorylation targeted to serine/threonine residues. The Y490 and Y785 of the receptor play particularly important roles in these events and stimulate the activation of three main pathways: ERK1/2 via Ras, phosphatidyl inositol - 3- kinase (PI3K) and PLC. However, a much broader array of phosphorylations is known to occur in both resting and stimulated cells. The aim of this study is to determine the profile of serine/threonine phosphorylation targets after stimulation of the TrkA receptor for 20 and to characterize the precise involvement of Y490 and Y785 in these processes. To avoid the stimulation of the second receptor of NGF (p75) and endogenous TrkA receptors, we used chimeric receptors composed of the ectodomain of the platelet-derived growth factor (PDGF) receptor and the transmembrane and endodomain of the TrkA receptor (denoted PTR), stably transfected into PC12 cells. The cells, grown in a medium containing heavy/light lysine/arginine (SILAC), were stimulated for 20 minutes with PDGF and lysed. The tryptic digest of proteins was enriched for phosphopeptides by a TiO2 column, further fractionated by strong cation exchange chromatography and analyzed by LC-MS/MS. The analysis of the phosphoproteome in cells expressing PTR showed quantitative changes in some previously known entities in signaling pathways, proving the specificity of the method, as well as many new modified entities. Interestingly, a comparison with a data set from HeLa (human) cells stimulated for the same time period by EGF (Olsen et al, 2006), showed a similar profile of protein kinase activation as judged by substrate specificity motifs. Analyses of cells expressing a PTR Y490F mutant and a PTR Y490/785F double mutant, stimulated under the same conditions, revealed three major classes of modifications: those dependent on Y490, those dependent on Y785 and those not dependent on either. This last group could be activated through the phosphorylation of one or more of the five other endodomain tyrosines of TrkA known to be phosphorylated but not previously identified as a docking/effector binding site(s). These activations are presumably not required for neurite proliferation but are likely involved in other functions stimulated by NGF. This work was supported by NIH NCRR grant P41 RR001614.
49/381
[O3] Quantifying Protein-Protein Interactions Within Noncovalent Complexes Using Electrospray Ionization Mass Spectrometry
Elisabetta Boeri Erba1, Konstantin Barylyuk1, Yang Yang1, Renato Zenobi1
1
Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland Contact: Elisabetta Boeri Erba (eboerierba@gmail.com)
Keywords: Proteins, peptides and small molecules quantification Several electrospray-mass spectrometry (ESI-MS)-based methods are available for determining the equilibrium association constant (Ka) between a protein and a small ligand, but current MSbased strategies are inadequate for measuring Ka of protein-protein interactions with accuracy. We expanded the application of ESI-MS-based titration to determine the strength of noncovalent interactions between proteins, forming a complex. Taking into account relative response factors (probability of being ionised, transmitted and detected), we determined Ka values of an equilibrium between dimers and tetramers at three different pH values (6.8, 3.4 and 8.4). We investigated the association of the lectin concanavalin A, whose dimer-tetramer equilibrium is affected by in-solution concentration and by pH. To calculate the constants of association (Kd) in solution, we also utilised isothermal titration calorimetry (ITC) for a comparison with MS-based titration method. At pH= 6.8 and pH= 8.4 the Ka values measured by MS and by ITC were in agreement. At pH= 3.4 we were able to measure the Ka only by MS, but not by ITC due to limited sensitivity of calorimetry. Our investigation illustrates the great potential MS for calculating the binding strength of protein-protein interactions within noncovalent complexes. The main advantages of MS over ITC are its sensitivity (i.e. the required amount of sample is >100 times less than the one necessary for ITC) and the possibility to obtain precise information on composition of protein complexes, their stoichiometry, their subunit interactions and their assembly pathway. Moreover, our study shows the strong influence of response factors on determining accurate protein-protein association constants.
50/381
[O4] Structure Determination of Complex Plant Glycosphingolipids by Tandem Mass Spectrometry

Corinne Bur1, Jean-Luc Cacas2, Fen Wang2, Karen Gaudin3, Frdric Domergue2, Sbastien Mongrand2, JeanMarie Schmitter 1
1 2 3
CBMN UMR5248 - CGF, 146, rue Lo Saignat, 33076 Bordeaux LBM UMR5200, 146, rue Lo Saignat, 33076 Bordeaux EA 4575 Universit Bordeaux Segalen, 146, rue Lo Saignat, 33076 Bordeaux
Contact: Corinne Bur (c.bure@cbmn.u-bordeaux.fr)
Keywords: Systems biology Glycosyl-Inositol-Phospho-Ceramides (GIPC) are the main sphingolipids of plant tissues, but the function of this complex family of compounds remains largely unknown. Therefore, the structural characterization of GIPC represents a critical step toward the understanding of their physiological function. In this work, GIPCs have been purified from plants cells (A. thaliana and N. tabacum cv. Bright Yellow 2, i.e. BY2), yielding multiple molecular species that have been analyzed by tandem mass spectrometry, using a combination of MALDI- and ESI-MS/MS. This method should open new avenues to decipher structurefunction relationships between glycosphingolipids and membrane organisation. GIPC extracts were analyzed by MALDIMS/MS in negative ion mode, using 2,6-dihydroxyacetophenone (DHA) as a matrix (MALDI Q-ToF Premier, Waters). ESI-MS/MS analyses were performed with a 5500 QTRAP (AB Sciex) instrument. Analyses performed by MALDI mass spectrometry provide a quick overview of the variety of GIPC structures found in a given type of plant cell. A comparison between several organisms is straightforward, using MS/MS to confirm the identifications. The ceramide part of identified GIPCs merely contains tri-hydroxylated long chain bases (t18:0 and t18:1) to which alphahydroxylated very long chain fatty acids are amidified, whereas the polar head part displays more diverse structures. MALDI mass spectra of A.thaliana and BY-2 cell samples revealed the occurrence of several clusters of GIPCs that can be grouped in six series (A to F) which differ by their mass range. For A.thaliana and BY-2 cells, each series was composed by several GIPC species differing by 2, 14 and 16 Da, corresponding to different degrees of unsaturation, carbon number or hydroxylation of the fatty acid chain. Fatty acid chains spent from 20:0 to 27:0, and from h20 to h26 for hydroxylated species. Mass differences between series were accounted by the number of saccharide units, and were either 162 Da (hexose) or 132 Da (pentose). Thus, MALDI-MS revealed that these plant GIPC structures had an increasing number of saccharide units, from two (series A) to seven (series F). The major compounds belong to series A for both A.thaliana and BY-2 cells. This result is in agreement with the previous report of hexose(R1)-hexuronic acid-inositol-phosphoceramide as major species in A. thaliana and N. tabacum leaves [1], where R1 is a hydroxyl group and an amine or an acetylamine, respectively. Further ESI-MS/MS experiments were conducted to confirm the structure identifications. Fragmentations observed in MALDI-MS/MS (singly charged ions) and ESI-MS/MS (doubly charged ions) were complementary for in-depth structure characterization of individual GIPCs among A-F series. Finally, up to 51 and 121 molecular species could be detected in A. thaliana and tobacco BY-2 cells, respectively. [1] J.E. Markham et al. J. Biol. Chem. 2006, 281, 22684.
51/381
[O5] Compatibilit entre histologie et imagerie par spectromtrie de masse ToFSIMS : dtection et cartographie de lipides au sein de tissus biologiques
Claudia BICH1, Sara VIANELLO2, Vincent GUERINEAU1, David TOUBOUL1, Sabine DE LA PORTE2, Alain BRUNELLE1
1
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
2
Centre de Recherche de Gif, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie du Dveloppement, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Contact: Claudia BICH (claudia.bich@icsn.cnrs-gif.fr)
Keywords: Imaging mass spectrometry Lhistologie permet de diffrencier simplement et rapidement sur un tissu biologique diffrentes structures (noyau, membrane) grce lutilisation de colorants spcifiques. Par exemple, le Rouge Nil permet de colorer les lipides neutres. Nanmoins aucune information molculaire dtaille nest obtenue par cette technique. Limagerie par spectromtrie de masse (ISM), de par la possibilit dobtenir simultanment un grand nombre dimages, semble tre une technique complmentaire lhistologie. Jusqu maintenant, il tait ncessaire dutiliser des coupes adjacentes pour lhistologie et lISM afin dtablir des parallles dans les rsultats et den tirer des conclusions pertinentes. Pourtant, il est parfois difficile dobtenir des coupes adjacentes adaptes pour les analyses. La possibilit de raliser lhistologie et limagerie par SM sur la mme coupe de tissu biologique a t lobjet de cette tude. Limagerie par spectromtrie de masse dions secondaires couple un analyseur temps de vol (ToF-SIMS), qui ne ncessite aucun prtraitement de lchantillon et qui nest pas destructive, sest rvle trs adapte pour cette tude. Des techniques de coloration de routine, telles que lHmatoxyline-Eosine (HE) ou le Rouge Nil (NR) ont t appliques sur des coupes de tissus biologiques avant ou aprs localisation de lipides par ToF-SIMS. Les coupes de 16 m dpaisseur ont t ralises laide dun cryostat CM3050-S -20C, dposes sur des lames en verre puis sches brivement sous vide. Certaines de ces lames ont t analyses par ToF-SIMS puis colores et analyses de nouveau par ToF-SIMS, dautres ont t directement colores puis analyses. Les images ioniques 500 m x 500 m (2 m de rsolution spatiale) ont t acquises avec un spectromtre de masse ToF-SIMS IV en modes dionisation positif et ngatif. Dans le cas des coupes colores puis analyses par ToF-SIMS, une dlocalisation de certains ions tels que le cholestrol (m/z 369 ion fragment caractristique en mode dionisation positif et m/z 385 en mode dionisation ngatif) a t observe aprs coloration par HE et NR. Sur les coupes de cerveau de rat, les images ont montr que le cholestrol tait toujours majoritaire au sein du corps calleux mais semble tre anti-co-localis aprs coloration. Au contraire, les images de ces mmes ions aprs les lavages au tampon phosphate (une des tapes) ne montrent aucune dlocalisation, et restent similaires aux images contrles. De plus, les rsultats prliminaires ont montr que lanalyse par ToF-SIMS nempche pas la coloration des coupes par la suite. Enfin, il a t possible de refaire une analyse de la surface par ToF-SIMS sur ces mmes coupes pour valuer la dlocalisation et le rendement dmission ionique secondaire. En conclusion, il est possible dobtenir les informations histologiques de coupes pralablement tudies par spectromtrie de masse ToF-SIMS.
52/381
[O6] ETD pression atmosphrique

David Touboul1, Alexandre Giuliani2, Julie Allegrand1, Olivier Laprvote4, Alain Brunelle1
1
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, Avenue de la Terrasse, 91198 Gifsur-Yvette
2 3 4
Ligne DISCO, Synchrotron Soleil, L'Orme des Merisiers, 91192 Gif-sur-Yvette Cepia, Institut National de Recherche Agronomique, BP 71627 , 44316 Nantes Chimie Toxicologie Analytique et Cellulaire, EA4463, Universit Paris Descartes, 75006 Paris
Contact: David Touboul (touboul@icsn.cnrs-gif.fr)
Keywords: Instrumentation, Organic and inorganic mass spectrometry, Signaling, interactomics and post-translational modifications Il est fondamental pour des tudes protomiques de possder des outils efficaces de fragmentation des peptides, afin daccder leur squence et leurs ventuelles modifications post-traductionnelles (MPT). De nombreuses techniques cohabitent actuellement. La dissociation induite par collision couple une source electrospray permet dobtenir de bons rendements de fragmentation mais nautorise pas le positionnement de certaines MPTs. linverse, le transfert lectronique dissociatif (ETD) permet lanalyse de MPTs labiles mais les rendements de fragmentations sont trs faibles. Le but de ce travail est de dvelopper de nouveaux outils pour la fragmentation efficace de peptides modifis ou non. Le principe gnral du transfert lectronique dissociatif pression atmosphrique est bas sur lutilisation de la photoionisation pression atmosphrique (APPI). Le peptide en solution est tout dabord transfr en phase gazeuse grce une source de type thermospray puis irradi par un faisceau intense de photons UV issu du synchrotron SOLEIL. Le rayonnement UV peut ioniser le solvant si lnergie des photons est suffisante. Lavantage de ce couplage unique au monde est la possibilit de varier lnergie des photons entre 5 et 20 eV, alors que les photons mis classiquement par une lampe krypton ont des nergies fixes 10,0 et 10,6 eV. Deux types dexpriences ont t mens : lune consiste faire varier la composition des solvants photoionisables, lautre additionner une molcule facilement photoionisable, appele dopant. La premire srie dexprience a permis de dmontrer grce des balayage en nergie que pour des photons dnergie suprieure au potentiel dionisation de lthanol, de lisopropanol ou du butanol, un transfert dlectrons entre les molcules de solvant vers les ions multichargs de la substance P est observ conduisant la formation en source dions fragments de type c, b et y, et la disparition totale des ions multichargs. Cette raction peut tre compare celle obtenue par ETD mais avec un rendement bien plus important. Dans le cas du mthanol, cette raction est par contre peu efficace et seul un transfert de proton entre le solvant et la substance P est observ. La seconde srie dexpriences a permis de dmontrer que des fragments intenses de type c, b et y sont aussi obtenus lorsque les photons possdent une nergie suprieure au seuil dionisation du dopant. Lanalyse dun peptide myristoyl a permis de confirmer ces phnomnes et la MPT a bien t localise en N-ter grce une srie presque complte dions de type c. Finalement lanalyse dun peptide phosphoryl a conduit la formation prfrentielle dune srie complte dions de type b-H3PO4. . La suite de ce travail consistera dvelopper un systme de nbulisation peu chauff afin de minimiser les fragmentations thermiques successives tout en contrlant les processus ETD pression atmosphrique.
53/381
[O7] Photo-SRM: laser photo-dissociation improves detection selectivity of selected reaction monitoring mode
Quentin ENJALBERT1, Romain SIMON2, Arnaud SALVADOR2, Rodolphe ANTOINE1, Sebastien REDON3, Florence DARBOUR5, Stephane CHAMBERT3, Yann BRETONNIERE5, Philippe DUGOURD1, Jrme LEMOINE2
1
LASIM, Universit Lyon 1, CNRS, Domaine Scientifique de la Doua Universit Claude Bernard Lyon 1 Btiment Alfred Kastler, 69622 Villeurbanne
2
LSA, Universit Lyon 1, CNRS, Domaine Scientifique de la Doua Universit Claude Bernard Lyon 1 Btiment CPE, 69622 Villeurbanne
3
INSA-Lyon, Laboratoire de Chimie Organique et Bioorganique, d INSA-Lyon, Laboratoire de Chimie Organique et Bioorganique, bt. J. Verne, 20 Avenue A. Einstein, 69622 Villeurbanne
4
CBMS, Institut de Chimie et de Biochimie Molculaires et Supramolculaires, UMR5246 ; CNRS ; Universit Lyon 1 ; INSA ; CPE-Lyon, Bt. Curien, 43 Bd, 69622 Villeurbanne
5
Laboratoire de Chimie de lENS Lyon, , 46 alle dItalie, 69364 Lyon
Contact: Quentin ENJALBERT (quentin.enjalbert@lasim.univ-lyon1.fr)
Keywords: Proteins, peptides and small molecules quantification , Instrumentation Selected Reaction Monitoring (SRM) carried out on triple quadrupole mass spectrometers coupled to liquid chromatography has been a reference method to develop quantitative analysis of small molecules in biological or environmental matrices for years and is currently emerging as a promising tool in clinical proteomics. However, sensitive assays in complex matrices are often hampered by the presence of co-eluted compounds that share redundant transitions with the target species. In the present work we document for the first time the potential interest of substituting classical gas-collision activation mode by laser photodissociation to track chromophore-derivatized target peptide absorbing at 532 nm diluted in a whole plasma trypsin hydrolysate. We anticipate that this technique coined photo-SRM, combined with chromophores endowed with reactivity towards generic chemical groups, might significantly improve the limit of quantification of classical SRM-based assays. The present presentation focused on the development of a new technique dedicated to quantification, called photo-SRM, where non-discriminating collision-activated dissociation mode has been replaced by a more specific photo-dissociation process governed by the absorbing properties of a targeted compound. The first example photo-SRM experiments has been developed in-house on a conventional triple quadrupole mass spectrometer. Proof of principle of SRM-based monitoring by laser-induced dissociation instead of classical collision activated mode was carried out with the chromophore-derivatized-Oxytocin as a model molecule diluted in whole plasma trypsin hydrolysate. This preliminary investigation clearly demonstrates that both the selectivity and detection level may markedly be improved during SRM monitoring by replacing the classical mode of activation by gas collision by a photon excitation providing a judicious overlap between the absorbing properties of the target molecule and the excitation wavelength of the laser beam. The lack of cross contamination within the transition channel between the target molecule and co-eluted endogenous interferences results here in a 50-fold improvement of the limit of quantification. The question rises now about how photo-SRM might be further optimized and extended to molecules that do not exhibit natural or specific light absorption. Novel aspect: First demonstration of Photo-SRM tool for analytical quantification.
54/381
[O8] Reflectron Positive ion Mode Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Sulfo-peptides
Sonia CANTEL1, Luc Brunel1, Keiichiro Ohara1, Christine Enjalbal1, Jean Martinez1, Jean-Jacques Vasseur1, Michael Smietana1
1
Institut des Biomolcules Max Mousseron (IBMM) UMR 5247 CNRS-Universit Montpellier 1 et Universit Montpellier 2, Universit Montpellier 2, Place Eugne Bataillon, 34095 Montpellier Contact: Sonia CANTEL (sonia.cantel@univ-montp2.fr)
Keywords: Signaling, interactomics and post-translational modifications Protein identification and characterization, has become one of the central activities in proteomics. The biological activity of many proteins is regulated by the extent and positions of posttranslational modifications (PTMs). More than 200 different PTMs have been described but only a minor fraction of those has been studied in detail. Indeed, most of these PTMs are lacking appropriate analytical methods that allow the sensitive, residue-resolved detection and localization of the modifications. It appears that sulfation of tyrosine is a PTM occuring almost exclusively on secreted and transmembrane spanning proteins. Evidence suggests up to 1% of all tyrosine residues of the total protein content in an organism can be sulfated. Mass spectrometry has been developed as a key technology for protein modification analysis and many studies on sulfopeptides are MALDI-based. It has been reported that the sulfo-moiety from sulfopeptides is readily and quantitatively lost in positive ion mode. Alternatively, MALDI ion negative mode has proven to be useful for analysis of sulfopeptides as the lability of deprotonated anionic sulfomoieties is lower than protonated neutral ones. Here we describe a peptide/small molecule non covalent interaction system allowing detection of mono- and polysulfated peptides in reflectron/linear positive mode by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). We will highlight strengths and limitations of this strategy and phospho-peptides detection approach will be discussed.
55/381
[O9] Dveloppement dune mthode de mtabolomique associant la RMN et lUHPLC-HRMS

Emilien JAMIN1, Ccile CANLET1, Maria-Hlna MEIRELES1, Marc DUBOIS1, Marie TREMBLAY-FRANCO1, Nicolas CABATON1, Fabien JOURDAN1, Daniel ZALKO1, Laurent DEBRAUWER1
1
INRA, UMR1331 TOXALIM, plateforme MetaToul, 180 Chemin de Tournefeuille, F31027 Toulouse
Contact: Emilien JAMIN (jamin@toulouse.inra.fr)
Keywords: Systems biology, Separative analysis methods, Organic and inorganic mass spectrometry La RMN et la spectromtrie de masse sont deux outils complmentaires en mtabolomique. Alors que la RMN est dsormais largement reconnue dans le domaine de la mtabolomique de par sa robustesse, la spectromtrie de masse est quant elle en pleine expansion, en particulier depuis larrive des colonnes chromatographiques de granulomtrie faible et larrive de la spectromtrie de masse trs haute rsolution de type Orbitrap. Dans ce travail, nous nous sommes intresss dvelopper une mthode associant en parallle la RMN et le couplage UHPLC-HRMS, qui a t applique une tude des effets du bisphnol A faibles doses au niveau cellulaire (cellules HepaRG). Nous nous sommes particulirement intresss loptimisation des paramtres de retraitement des donnes de spectromtrie de masse par le pack XCMS. Des cellules HepaRG en culture ont t exposes ou non des faibles doses de bisphnol A (DMSO versus BPA 10e-6, 10e-9 et 10e-12mol/L). Les extraits cellulaires ont ensuite t analyss tout dabord par RMN du proton (600MHz, Bruker) puis par couplage UHPLC-FTMS, constitu dun systme RSLC3000 (Dionex), dun gradient de phase mobiles composes de H2O/mthanol/acide actique 95/5/0,1 et mthanol/acide actique 100/0,1 lues 0,3 mL/min, et dune colonne chromatographique Hypersil Gold (100x2,1 mm, 1,9 m) (Thermo Scientific) 30C. La dtection a t ralise avec un spectromtre de masse LTQ-Orbitrap XL (Thermo Scientific) en mode FTMS avec une source dionisation Electrospray fonctionnant en mode positif ou ngatif. Tout comme les autres logiciels de traitement de donnes LC-MS de mtabolomique, le pack XCMS utilise deux grandes fonctions : la dtection/intgration de pics chromatographiques au sein des signaux bruits, et le regroupement/alignement des variables (couple m/z et tR) entre les chantillons. Ces deux fonctions sont bases sur diffrents algorithmes prsents dans le pack XCMS, mais qui ncessitent le rglage de paramtres la fois nombreux et peu explicites. Une interface dveloppe au laboratoire (MetMS) rend le paramtrage de XCMS plus ais depuis limportation des fichiers, jusqu la cration de la liste des pics dtects, par lutilisation de fentres de commande. De plus, cette interface permet de visualiser les spectres de masse et les chromatogrammes chaque tape du traitement XCMS. Ainsi, nous avons optimis chaque paramtre XCMS nos conditions danalyses, par ltude de la dfinition de ces paramtres et leur influence sur la dtection de pics. Les donnes ainsi gnres par RMN et UHPLC-HRMS ont t traites par des mthodes statistiques multivaries. La rgression PLS-DA a permis une bonne sparation des diffrents groupes. Lensemble du processus mis en place depuis le traitement des chantillons jusquau traitement des donnes produites sera prsent en sappuyant sur lapplication traite.
56/381
[O10] Prises dempreintes urinaires par LC-HRMS : La mtabolomique, une stratgie innovante pour le dpistage de lutilisation danabolisants en levage.
Sylvain Chreau1, Frdrique Courant1, Fabrice Monteau1, Jean-Philippe Antignac1, Gaud Dervilly-Pinel1, Bruno Le Bizec1
1
LABERCA, ONIRIS, Route de Gachet site de la chantrerie, 44307 Nantes, France
Contact: Sylvain Chreau (sylvain.chereau@oniris-nantes.fr)
Keywords: High mass and high resolution mass Spectrometry, Separative analysis methods, Bioinformatics et biostatistics dedicated to proteomics Les caractres globaux et non cibls dune tude mtabolomique en font une approche de choix dans de nombreux domaines. Applique la scurit chimique des aliments, la mtabolomique laisse entrevoir de nombreuses perspectives comme, par exemple, lambition de dcouvrir de nouveaux biomarqueurs rvlateurs dune contamination de la (des) denre(s). Dans ce contexte, et en raison de luniversalit de la dtection et des niveaux de sensibilit atteints, la spectromtrie de masse sest naturellement impose comme lune des technologies de choix pour la prise dempreintes mtabolomiques. Dans le cadre de ses recherches appliques la dtection des pratiques frauduleuses en levage, le LABERCA a dvelopp depuis plus de 5 ans une stratgie complte de prise dempreinte mtabolomique (LC(C18)-ESI-Orbitrap(HRMS) // XCMS // SIMCA P+) afin de rvler de potentiels candidats biomarqueurs dun traitement anabolisant. Applique diffrentes familles de composs (Hormone de croissance, strodes, -agonistes), lapproche dores et dj fait ses preuves [1-8] puisque de robustes modles statistiques permettant de discriminer des groupes danimaux (i.e : contrles & traits) ont t tablis et lidentification structurale, tape critique et dlicate, a pu tre ralise pour certains candidats biomarqueurs. Lobjectif est prsent de dvelopper un outil de dpistage bas sur le suivi cibl des biomarqueurs identifis, par le dveloppement de mthodes quantitatives par spectromtrie de masse (UPLCMS/MS). Il sagit en particulier de travailler loptimisation de la dtection de ces composs, par lvaluation de techniques chromatographies alternatives (HILIC) ou encore laide dune quantification plus fine (dilution isotopique). Enfin un modle combinant les concentrations de chacun des candidats biomarqueurs permettra de valider lapproche et la pertinence de leur suivi pour mettre en vidence une suspicion de pratique anabolisante en levage. [1] Courant F., Pinel G. et al., Analyst, 2009, 134:1637-1646. [2] Kieken F., Pinel G. et al., Anal Bioanal Chem, 2009, 394:2119-2128. [3] Anizan S., Bichon E. et al., J Chrom A, 2010, 1217:6652-6660. [4] Pinel G., Mooney M. et al., TrAC, 2010, 29:1269-1280. [5] Kieken F., Pinel G. et al., Metabolomics, 2011, 7:84-93. [6] Antignac J.P., Courant F. et al., TrAC, 2011, 30:292-301. [7] Pinel G., Weigel S. et al., Anal. Chim. Acta, 2011, in press. [8] Anizan S., Di Nardo D. et al., Anal. Chim. Acta, 2011, in press. Link: http://www.laberca.org
57/381
[O11] Mechanisms leading to unusual ECD fragments: the ion spectroscopy perspective.
Edith NICOL1, Julia CHAMOT-ROOKE1, Guillaume VAN DER REST1
1
Laboratoire des Mecanismes Reactionnels, Ecole Polytechnique, CNRS, Route de Saclay, 91128 Palaiseau, France Contact: Edith NICOL (edith@dcmr.polytechnique.fr)
Keywords: Organic and inorganic mass spectrometry As introduced by Roman Zubarev et al. in 1998, ECD has been described as leading mostly to c and z fragment types [1]. The observation of other fragment types (a, b, y, w) has also been described in the literature, but often as either secondary fragmentation of the major c/z ions, or as secondary or minor processes. Recent experimental work in our group on a pentapeptide series (AGXLK - X=A, D, E, S, W) of ions, as well as analysis of a database of 11 000 ECD spectra from doubly charged tryptic peptides [2] revealed that these pathways can represent the major ones for small (less than 9 residues) size peptides. Therefore, far from being an epiphenomenon, observed only for some very specific peptides, these fragmentation pathways can truly be observed in the ECD fragmentation of peptides present in bottom-up proteomics studies. The database analysis not only revealed the extent of these atypical fragmentation behaviors, but also led to question assumptions on the fragmentation pathways leading to these ions. For instance, the w fragmentation pathway has usually [3] been ascribed to a secondary fragmentation of z ions, through a side-chain loss. Our database analysis shows that the w ions do not arise from the major z ions, therefore leading to the question of the mechanism accounting for their formation. No alternative mechanism is present in the literature to this date. A similar question arose for b ions formed by ECD, for which at least three pathways are proposed in the literature. The structure of these fragments could provide hints on the mechanistic pathways followed to reach these products. In light of the questions raised above, infra-red action spectra of some w and b ECD fragments were recorded at the CLIO facility in Orsay. Briefly, ions of interest are produced from ECD fragmentation of selected precursors in the cell of an FT-ICR mass spectrometer and irradiated by a tunable infra-red free-electron laser beam. IRMPD fragmentation efficiency is recorded as a function of wavelength. Theoretical reference spectra for series of possible structures were produced through DFT geometry optimizations and frequency calculations and compared with the experimental spectra. For b ions, the IR action spectra of the corresponding CID fragments were also recorded and used for comparison. This work led us to obtain the first IR spectra of w type ions, which seem to have the expected linear ketene structure. For b ions, some ions display a different experimental spectrum for the ECD fragments compared to the CID fragments, which would point out to a different formation mechanism. [1] Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W., J Am Chem Soc 1998, 120, 3265. [2] Van der Rest, G.; Hui, R.; Frison, G.; Chamot-Rooke, J., J. Am. Soc. Mass Spectrom. 2011, in press. [3]Savitski, M. M.; Nielsen, M. L.; Zubarev, R. A., Anal. Chem. 2007, 79, 2296.
58/381
[O12] Investigating the unusual behavior of Metolachlor under chemical ionization in hybrid ion trap mass spectrometry
Stphane Bouchonnet1, Pierre-Henry Goulden1, Christophe Genty1, Sophie Bourcier1, Michel Sablier1
1
Laboratoire des Mcanismes Ractionnels, Ecole Polytechnique, 91128 Palaiseau
Contact: Stphane Bouchonnet (stephane.bouchonnet@dcmr.polytechnique.fr)
Keywords: Organic and inorganic mass spectrometry, Instrumentation, Proteins, peptides and small molecules quantification Acetochlor, Alachlor and Metolachlor are widely used herbicides frequently detected in river waters. Known as potential endocrine disruptors, their toxicity for human has been clearly established. The characterization of their ozonation and photolysis by-products has been the subjects of recent investigation since some of them could also been responsible for endocrine disruption. The structural elucidation of by-products is usually performed using LC-MS/MS and GC-MSn analysis for polar and little or not polar compounds, respectively. In GC-MS, the use of chemical ionization (CI) is expected to provide abundant MH+ ions allowing direct access to the molecular weight of the analyte while electron ionization (EI) provide abundant fragment ions but only trace amounts of M+. ions for the herbicides of interest. We have been recently puzzled by the strange behavior of Metolachlor which, unlike the other mentioned pesticides, provides a very abundant monochlorinated ion at m/z 295/297 under ammonia positive chemical ionization using an hybrid ion trap mass spectrometer in GC-MS coupling. This ion observed only at trace amounts when using methanol as reagent gas. It disappears when attempting to isolate it to perform MSn experiments. Curiously, this ion at m/z = M+12 is not observed for the herbicides Acetochlor and Alachlor which present very similar chemical structures. The chemical structures of the m/z 295 and m/z 297 ions and the explanation of the observed phenomenon were elucidated on the basis of experiments including isotopic labeling and modifications of the operating conditions of the ion trap mass spectrometer. This work allows to give new recommendations for an optimized use of hybrid source ion trap mass spectrometers.
59/381
[O13] Characterization of cellular proteasome complexes diversity by proteomic approaches

Bertrand Fabre1, Bernard Monsarrat1, Marie-Pierre Bousquet-Dubouch1, Odile Burlet-Schiltz1
1
Institut de Pharmacologie et Biologie Structurale, IPBS, CNRS, 205 route de Narbonne, 31077 Toulouse, France
2
Universit de Toulouse, 118 route de Narbonne, 31077 Toulouse
Contact: Bertrand Fabre (bertrand.fabre@ipbs.fr)
Keywords: Signaling, interactomics and post-translational modifications, Proteins, peptides and small molecules quantification Proteasome is a multimeric protease complex, which degrades more than 80% of proteins in the cell. Due to its substrate wide range, proteasome is involved in the regulation of many important cellular processes (cell division, transcription, cell differentiation). Proteasomes complexes consist of a 20S catalytic core particle comprising 14 to 17 different proteins assembled in four stacked ring-shape heptamers. This core complex can be associated to one or two regulatory particles of identical or different protein composition and can also recruit other so-called Proteasome Interacting Proteins (PIPs). Proteasome activity is tightly regulated by the nature of these associated proteins. Proteasome complexes are present in the cytoplasm (free, associated with cytoskeletal elements or bound to the endoplasmic reticulum) and in the nucleus but little is known about the distribution of the different associated regulators in each cell compartment. To answer this question, we have developed an affinity purification-quantitative mass spectrometry strategy using a 20S proteasome core particle subunit as bait. This allows the enrichment of all combinations of 20S-regulator endogenous proteasome complexes in the cell. To stabilize the labile association between the 20S core particle with its regulators and PIPs, we have performed in vivo cross-linking of protein-protein interactions using formaldehyde [1]. Formaldehyde was introduced very early in the proteomic workflow, so that real biological events could be frozen. Optimization of formaldehyde concentration was performed to maximize the specific activity of purified proteasomes, the yield of purification and the recovery of regulators and PIPs. The fractionation protocol was also optimized to deal with the cross-linked cells and to obtain proteasome complexes specific of the cytoplasm, the microsome and the nucleus compartments. A label-free quantitative proteomic workflow [2] was then performed to compare proteasome complexes diversity in each cellular compartment and in different leukemia cell lines exhibiting differences in proteasome activity. Determining the composition in regulators of the proteasome, according to the cellular localization, will allow a better understanding of their functions. 1 Bousquet-Dubouch MP, Baudelet E, Guerin F, et al. Affinity purification strategy to capture human endogenous proteasome complexes diversity and to identify proteasome-interacting proteins. Mol Cell Proteomics 8, 1150-1164 (2009). 2 Mouton-Barbosa E, Roux-Dalvai F, Bouyssie D, et al. In-depth exploration of cerebrospinal fluid by combining peptide ligand library treatment and label-free protein quantification. Mol Cell Proteomics 9, 1006-1021 (2010).
60/381
[O14] Optical profiling of an Agilent Jet Stream Technology electrospray by LaserInduced-Fluorescence coupled to mass spectrometry measurements
Marion Girod1, Xavier Dagany1, Rodolphe Antoine1, Philippe Dugourd1
1
Laboratoire de Spectromtrie Ionique et Molculaire; C.N.R.S. et Universit Lyon I, Bat. A. Kastler, 43 bd du 11 Novembre 1918, 69622 Villeurbanne Cedex - FRANCE Contact: Marion Girod (mgirod@lasim.univ-lyon1.fr)
Keywords: Organic and inorganic mass spectrometry, Instrumentation La spectromtrie de masse aprs ionisation electrospray (ESI) est devenue une des techniques analytiques les plus utilises et consiste former des ions intacts en phase gazeuse par lectronbulisation dun analyte en solution. Cependant, les mcanismes mis en jeu lors de cette ionisation sont relativement peu connus, en particulier ceux du processus de dsolvatation au sein de la plume entre l'metteur electrospray et le capillaire dentre du spectromtre de masse. Des mesures spectroscopiques peuvent aider tudier les proprits chimiques et physiques des gouttelettes de la plume electrospray ainsi que les proprits des ions au sein de ces gouttelettes. Un montage exprimental permettant de raliser des mesures de fluorescence au sein de la plume electrospray a t dvelopp, tandis que des spectres de masse danalytes sont enregistrs simultanment. Des images 2D de la plume electrospray ont t obtenues en mesurant la concentration (c.--d. le signal de fluorescence) d'une solution de colorant rhodamine 6G (Rh6G) avec une rsolution spatiale denviron 200m. Afin de sonder les proprits physiques et chimiques des gouttelettes au sein de la plume ESI, des colorants chromiques ont t utiliss. L'intensit de fluorescence ou la longueur d'onde de diffrents colorants chromiques est corrle avec les proprits chimiques et physiques, en utilisant les courbes d'talonnage tablies. Les spectres d'mission du Nile Red (NR) dans un mlange d'actonitrile/eau ont t enregistrs afin de dterminer la composition de solvant dans la plume pour diffrents paramtres de spray. Les rsultats indiquent clairement que la composition du solvant dans les gouttelettes n'est pas homogne dans la plume ESI, en raison de l'vaporation des solvants. La fluorescence induite par laser a galement t employe pour profiler des changements de pH lors de lvaporation des gouttelettes dans la plume ESI en mesurant des spectres d'mission du colorant pHchromic C.SNARF-1. Ce couplage de mesures de spectromtrie de masse et de spectroscopie optique permet d'tudier la relation entre l'tat de charge observ d'anions de peptide et les changements de pH au sein du spray en fonction des paramtres de source tels que le dbit et la temprature du gaz de gainage qui jouent sur le rendement dvaporation de gouttelettes.
61/381
[O15] Tabagisme actif/tabagisme passif, une tude diffrentielle de la fraction particulaire

Sbastien Schramm1, Vincent Carr1, Jean-Luc Scheffler 2, Frdric Aubriet1
1 2
Laboratoire de spectromtrie de masse et de chimie laser, 1 boulevard Arago, 57078 Metz, France Laboratoire Ascal, Parc d'activit Forbach Ouest, 57600 Forbach
Contact: Sbastien Schramm (schramm@univ-metz.fr)
Keywords: High mass and high resolution mass Spectrometry, Organic and inorganic mass spectrometry La fume de cigarette est un contaminant majeur de lair intrieur (indoor). De nombreux constituants de ces fumes, essentiellement gazeux, ont t identifis. Certains prsentent des proprits toxiques, carcinognes et/ou mutagnes. Outre ces espces gazeuses, les fumes de cigarette contiennent une fraction particulaire importante. Les agences sanitaires et de sant sont actuellement proccupes par lexamen de ces particules. Celles-ci possdent non seulement des temps de sjour levs dans lorganisme mais aussi une composition complexe et varie et sont le sige de ladsorption dun grand nombre de molcules issues de la combustion de la cigarette. A la diffrence de la pollution outdoor pour laquelle des normes existent en termes de contaminants particulaires (PM10 voire PM2.5), en atmosphre confine aucune lgislation nest encore propose. Dans lobjectif daccder la composition dtaille des particules de fumes de cigarettes, nous nous sommes intresss plus particulirement la caractrisation et la comparaison des fumes inhales par le fumeur (MSS), celles manant de la cigarette entre deux bouffes (SSS), et les fumes expires par le fumeur (EXS), ces deux dernires fractions tant celles auxquelles est expos lindividu dans le cadre du tabagisme passif. Aprs avoir valid en relation avec le laboratoire ASCAL les mthodes de prlvements par le suivi de traceurs spcifiques, les diffrents types de particules sont analyses par dsorption/ionisation laser couple la spectromtrie de masse FTICR. La haute rsolution et la prcision sur la mesure du rapport m/z permettent lattribution de faon non ambige dune formule brute aux 1000 2000 signaux observs dans la gamme m/z 150500. Lemploi de reprsentations spcifiques (carte de Kendrick et diagramme de Van Krevelen) assiste la comparaison des diffrents types de fumes. Les rsultats montrent que la nature et la composition des MSS sont significativement diffrentes de celles des SSS pour lesquelles des composs la fois moins oxygns et moins saturs sont observs [1]. Lexamen des EXS rvle quant lui peu de diffrences de composition entre les deux individus soumis ltude mais une diffrence importante lorsquelles sont compares aux MSS. De manire plus spcifique, il est constat un enrichissement en composs aromatiques tels que les HAP et une diminution de la quantit des composs les plus polaires. Des changes entre molcules adsorbes la surface des particules et milieu physiologique seraient mme dexpliquer ce comportement. [1] Schramm, S.; Carr, V.; Scheffler, J.-L.; Aubriet, F. Anal. Chem. 2011, 83, 133142.
62/381
[O16] Proteomic targeting of angiogenic cell lines by fumagillin mostly relies on the expression levels of METAP1
Frdric Frottin1, Imene Dridi1, Christelle Espagne1, Willy V. Bienvenut2, Thierry Meinnel1, Carmela Giglione1
1 2
CNRS, ISV, UPR2355, Btiment 23A, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette CNRS, ISV, FRC3115, Btiment 23A, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette
Contact: Willy V. Bienvenut (willy.bienvenut@isv.cnrs-gif.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications Fumagillin and its derivatives are therapeutically-useful compounds for their capacity to reduce cancer progression by blocking angiogenesis, a process which is required to feed the tumor and allow it to develop [1]. Fumagillin exerts indeed strong specificity for angiogenic cell lines such as HUVEC [2] and its most recent derivatives now display little neurotoxic side effects [3]. So far however, the primary molecular basis for this specificity are yet unknown. The specific molecular target of fumagillin is METAP2, one of the two METAP with METAP1 in the cytosol of animal cells [4]. METAPs are in charge of N-terminal Methionine Excision, an essential pathway of cotranslational protein maturation [5]. We have measured the cellular levels of both cytosolic METAP mRNAs in a number of cell lines and shown that cell sensitivity to fumagillin is correlated to METAP mRNA level, and particularly to that of METAP1. Indeed, when the METAP1 mRNA level is very low, as is the case of HUVEC and the glioblastoma cells U-87, the cells are very sensitive to fumagillin with IC50 values in the nanomolar ranges. If the METAP1 mRNA level is higher than that of HUVEC or U-87, the cells become insensitive to the drug (IC50 >100 M). Thus, we propose that the METAP1 expression can be routinely checked in several tumors and its level used as a prognosis marker to predict response to fumagillin and treatment including possible side effects. We are currently analyzing the direct impact of fumagillin treatment on protein N-termini of the whole proteome in HUVEC cells. After protein extraction, we specifically select N-terminal peptides and assess the global impact of inhibiting METAP on N-Methionine Excision. The data will be presented and discussed. References 1. Folkman & Ingber (1992) Semin. Cancer Biol. 3, 89-96. 2. Ingber et al. (1990) Nature 348, 555-7. 3. Satchi-Fainaro et al. (2004) Nat. Med. 10, 255-61. 4. Sin et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 6099-103. 5. Giglione et al. (2004) Cell. Mol. Life Sci. 61, 1455-74. Keywords: Angiogenesis, N-terminal modifications, cancer.
63/381
[O17] Ldi-ms of proteolytic synthetic model peptides deposited on silicon-based nanowires: an alternative to maldi pmf?
Mathieu Dupr1, Yannick Coffinier2, Rabah Boukherroub2, Sonia Cantel1, Jean Martinez1, Christine Enjalbal1
1 2
Institut des Biomolcules Max Mousseron (IBMM UMR 5247), Universit Montpellier 2, 34095 Montpellier
Institut dElectronique, de Micro-electronique et de Nanotechnologie (IEMN UMR CNRS 8520), Universit de Lille Nord de France, 59652 Villeneuve dAscq Contact: Mathieu Dupr (mathieu.dupre@univ-montp2.fr)
Keywords: Organic and inorganic mass spectrometry Matrix-free Laser Desorption/Ionization (LDI) mass spectrometry using specific inert surfaces to promote ion formation has been widely investigated the last decade.1 In addition to porous silicon through the original DIOS technique,2 different solid materials were tested as potent LDIpromoting agents, such as metals, carbon-based structures, porous particles, nanomaterials and more recently, ordered three-dimensional silicon architectures. Disposable ready-to-use NanoAssisted Laser Desorption/Ionization (NALDITM) target was also developed for matrix-free LDI analysis of small molecules and peptides.3, 4 Following our recent development of a straightforward low cost LDI method based on the use of porous chromatography materials for the analysis of peptides5, we explored other inert silicon/silica-based materials. Taking into account that nanostructured silicon based materials were providing good detection sensitivity and that surface derivatization has a great influence on the ionization yield, 6 we choose to evaluate the efficiency of LDI-MS carried out on functionalized Silicon Nanowires for the analysis of a large variety of model peptides designed in our laboratory. In this study, we focus on C18-Silicon Nanowires as matrix in LDI-MS for the analysis of small peptides mimicking proteolytic sequences (500-1700 Da). The detection of 30 peptides has been investigated on Nanowires prepared according to 3 different processes. We also realize a set of repeatability experiments on these peptides to assess the interspot and intraspot variations, representing about 800 MS analyses. Moreover, we characterize these peptides in MS/MS experiments and compare the fragmentation data with those obtained with sample conditioned in HCCA organic matrix. Finally, we carry out the analysis of various peptide mixtures in order to probe the potent spectral discrimination in LDI-MS and compare the results with the same experiment conducted with organic matrices in MALDI analyses. Through this set of experiments, we were able to assess the LDI performances in terms of sensitivity, repeatability and robustness of C18-Silicon Nanowires as ionizing surfaces within the framework of peptide analysis such as peptide mass fingerprinting in proteomics. [1] Peteron DS. Mass Spectrom. Rev. 2007, 26: 19-34. [2] Wei J, Buriak J, Siuzdak G. Nature 1999; 399: 243-246. [3] Gunin E, Lecouvey M, Hardouin J. Rapid Commun. Mass Spectrom. 2009; 23, 1395-1400. [4] Shenar N, Cantel S, Martinez J, Enjalbal C. Rapid Commun. Mass Spectrom. 2009; 23, 23712379. [5] Shenar N, Martinez J, Enjalbal C. J.Am.Soc.Mass Spectrom. 2008; 19: 632-644. [6] Piret, G. l.; Drobecq, H.; Coffinier, Y.; Melnyk, O.; Boukherroub, R. Langmuir 2009, 26 : 13541361.
64/381
[O18] Evaluation of label-free quantitative proteomic methods together with sample fractionation for the large-scale analysis of inflammatory endothelial cells
Anne Gonzalez de Peredo1, Violette Gautier1, Emmanuelle Mouton-Barbosa1, David Bouyssi1, Nicolas Delcourt1, Mathilde Beau1, Jean-Philippe Girard1, Odile Burlet-Schiltz1, Bernard Monsarrat1
1
Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 route de Narbonne, 31077 Toulouse, France
Contact: Anne Gonzalez de Peredo (gonzalez@ipbs.fr)
Keywords: Proteins, peptides and small molecules quantification , Bioinformatics et biostatistics dedicated to proteomics, Systems biology Many bioinformatic tools developed in recent years for the quantification of MS data in labelfree experiments use pattern-based methods and generation of LC-MS maps. Here, we applied the MFPaQ software, which uses an identity-based extraction approach, starting from peptide identification results to go back in the MS scans in order to retrieve peptide intensity values. In such label-free quantitative studies performed on complex protein samples, a compromise has to be found between reproducibility and high proteomic analytical coverage. The latter is often achieved through sample fractionation by 1D SDS-PAGE, which may induce experimental bias during the label-free comparison of samples processed and analyzed independently. We thus evaluated the efficiency of our label-free workflow with or without protein fractionation by 1D SDS-PAGE. To this aim, a whole-cell lysate of primary human endothelial cells was analyzed by nanoLCMS/MS, either in one analytical run, or after fractionation into 12 gel bands. Samples were run on an Orbitrap-Velos instrument with high sequencing speed in order to improve MS/MS sampling and analytical coverage. Technical gel replicates or LC-MS replicates were performed to evaluate variability and systematic errors due to upstream sample processing steps (electrophoretic migration, trypsin digestion, peptide extraction) or to final analytical steps (LCMS measurement and bioinformatic extraction of peptide XICs by the software). Data normalization and integration procedures were used in MFPaQ to correct LC-MS variability and errors related to non-reproducible electrophoretic migration of proteins in the case of sample fractionation. Our results show that for one-shot analysis, label-free quantification can be achieved with MFPaQ on about 700 proteins with good accuracy (median CV of 5%, 99% proteins with CVs<48%). Sample fractionation largely improved the depth of proteomic coverage (about 3600 proteins quantified), and this was associated with a moderate decrease of quantitative measurement reproducibility (median CV of 7%, 99% proteins with CVs<62%). The method was applied for the large-scale quantitative analysis of primary human endothelial cells stimulated by proinflammatory cytokines such as TNF, INF and IL1. It allowed us to identify and quantify about 5000 unique proteins, providing an in-depth analysis of the endothelial cell proteome and a detailed characterization of the proteomic variations associated with the inflammatory response. Link: http://proteomique.ipbs.fr/
65/381
[O19] Microorganism characterization for the clinics using SRM

Yannick Charretier1, Christine Franceschi2, Xavier Lacoux3, Gilles Zambardi2, Victoria Girard2, Sonia Chatellier 2, Arnaud Salvador4, Gaspard Gervasi1, Jrme Lemoine4, Jean-Philippe Charrier1
1 2 3 4
Recherche Technologique, bioMerieux SA, chemin de l'orme, 69280 Marcy L'Etoile R&D Microbiologie, bioMerieux SA, 3 Route De Port Michaud, 38390 La Balme Les Grottes R&D ImmunoEssais, bioMerieux SA, chemin de l'orme, 69280 Marcy L'Etoile
ANABIO, UMR 5180, CNRS / Universite de Lyon, Lyon-1 (UCBL-1), 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex Contact: Yannick Charretier (yannick.charretier@biomerieux.com)
Keywords: Proteins, peptides and small molecules quantification , Clinical proteomics Background: Recently, proteomics delivered its first clinical application in routine with identification of microorganisms using MALDI-TOF. However this technology, based on protein fingerprints, provides only a probability of identification and is not well suited for antibiotic susceptibility testing (AST) or virulence detection. To overcome MALDI-TOF limitations, we propose to use a triple-quadrupole mass spectrometer working in Specific Reaction Monitoring (SRM) mode, coupled with a conventional bore chromatography. Methods : Multiplexed SRM methods enabling an absolute identification of the microorganism as well as the determination of mechanisms of resistance, virulence and strain typing have been developed in our lab. After tryptic digestion either a colony or crude sample, is injected through a short chromatographic gradient (24 min) into a triple quadrupole mass spectrometer working in SRM mode. Results: As an example, methicillin-resistant Staphylococcus aureus (MRSA) strains could be fully characterized in 24 minutes using the multiplexed detection of 36 peptides from 14 proteins, including Penicillin-Binding Protein 2a (PBP-2a) and Panton-Valentine Leucocidin (PVL) toxin. Candida albicans yeast pathogenicity could be characterized, as well, by quantification of Ergosterol metabolite for resistance prediction and Lipase-8 protein for virulence estimation. Conclusions: Multiplexed and quantitative capabilities of SRM offer a unique possibility to develop targeted methods for characterization of microorganisms either after culture or directly on crude sample. The power of this state-of-the-art approach is the development speed, which one can adapt methods to capture the rapid microorganism evolution in biological mechanisms. This technology is expected to bring new proteomics applications into the clinic in the near future.
66/381
[O20] Targeted proteomics profiling of the plasma membrane transportome in Arabidopsis thaliana
Jean-Marc MONNEUSE1, Madeleine SUGANO1, Thierry BECUE1, Vronique SANTONI2, Sonia HEM1, Michel ROSSIGNOL1
1 2
Laboratoire de Protomique Fonctionnelle, INRA UR 1199, 2 place Viala, 34060 Montpellier cedex 1, France
Biochimie et Physiologie Molculaire des Plantes, INRA UMR 5004, 2 place Viala, 34060 Montpellier cedex 1, France Contact: Sonia HEM (hem@supagro.inra.fr)
Keywords: Proteins, peptides and small molecules quantification , Systems biology Plants are exposed to various and changing environmental conditions. With respect to the availability of nutrients, a well-established and nearly general response is the adaptation of the expression level of those transporters ensuring the uptake of the corresponding nutrient. A large part of them is located at the plasma membrane and a general feature is that they belong to multigene families with high homology between members. However, the high level of homology may hamper precise identification of isoforms and prevent their quantification. For the same reason, immunological approaches are often not effective presently for a number of isoforms. Beside shotgun strategies, however, dedicated approaches focusing on specific peptides and fragments are also emerging, but little used for sets of membranes proteins and never to date for plant membrane proteins from multigene families. In this work, we set up a targeted Multiple Reaction Monitoring (MRM) proteomics approach, based on the use of proteotypic peptides and isotopically labeled analogs, for the simultaneous identification and quantification of transporter isoforms in Arabidopsis plasma membrane. Focus was given to four families of transporters, the proton pumps of the AHA (Autoinhibited H+ATPases) family, the water channels of the PIP (Plasma membrane Intrinsic Proteins) subfamily of aquaporins, the ammonium transporters of the AMT1 sub-family and members of the NRT1 and NRT2 groups of nitrate transporters. In the strategy used, proteotypic peptides were selected firstly according to: their unicity in the whole Arabidopsis genome, their predicted potential ion-current response, their chromatographic behavior and their amino acid composition to avoid post-translational modification. Then, corresponding heavy forms of the 28 selected peptides were used for technical optimization (declustering potential, DP; collision energy, CE) using a stepwise procedure and LC-MS [1] and for quantification of the corresponding light version in biological samples. This workflow was shown to allow the analysis of isoforms in the fourth families, including as well major membrane proteins as transporters of lower abundance. Similarly, it was used to investigate the effect of a salt stress on the expression of these latter 20 transporters in roots and identify the responding isoforms. Such a resource (the peptides, their transitions and associated optimized detection conditions, as well as the synthetic peptides them-self) presents the advantage to be reusable and progressively expandable, thus opening the possibility of quantitative and isoform-specific investigation of the transportome in the model plant Arabidopsis.
[1] Lange, V., Picotti, P., Domon, B., Aebersold, R., Selected reaction monitoring for quantitative proteomics : a tutorial. Mol. Syst. Biol. 2008, 4, 222.
Link: http://www1.montpellier.inra.fr/proteome/index.htm
67/381
[O21] Effets de lactylation des peptides de la peau de la rainette Pachymedusa dacnicolor pour un squenage de Novo par MALDI-TOF/TOF
Claire lacombe1, Constance Auvynet3, Germain Tong1, Grard Bolbach1, Emmanuelle Sachon1
1 2 3
Universite P. et M. Curie Paris 6, UMR 7203 CNRS-UPMC-ENS, 4 place Jussieu, 75005 Paris, France Plateforme de spectromtrie de masse et protomique, IFR83, 7-9 quai saint bernard, 75005 Paris, France Piti salptrire, 91, Bd de l'hpital, 75013 Paris, France
Contact: Emmanuelle Sachon (emmanuelle.sachon@upmc.fr)
Keywords: Clinical proteomics Les grenouilles dAmrique latine synthtisent et scrtent au niveau de leur peau, des peptides biologiquement actifs. Dans cette tude, nous nous intressons aux peptides issus de la peau de la rainette, Pachymedusa dacnicolor, afin disoler et caractriser de nouveaux peptides dintrt thrapeutique. Pour ce faire, nous travaillons sur des exsudats de peau. Les peptides contenus dans ces exsudats sont spars par tamis molculaire suivi dHPLC. Les fractions obtenues sont testes pour leur activit biologique puis 4 fractions actives ont t analyses par spectromtrie de masse. Dans ces fractions, 6 peptides ont t identifis et fragments par MALDI-TOF/TOF (squenage de Novo). Trois dentre eux avaient dj t publis (DMS-DA5, DMS-DA6 et DMSDA8). Les trois autres peptides coexistent dans la quatrime fraction et possdent une structure primaire N-terminale identique (rsidus 1 29). Cette squence commune, qui avait t dduite partir de l'ADN de grenouille en 1998 est la suivante : ALWKTLLKKVGKVAGKAVLNAVTNMANQN-COOH. Les deux autres peptides de cette fraction possdent ces 29 rsidus +E et +EQ en C-terminal. Le peptide DMS-DA6 a t synthtis sous forme amide ou non en C-terminal. Lactivit antimicrobienne des deux peptides a t teste sur deux souches de bactries (Gram+ et -) et leur structure secondaire dtermine par dichrosme circulaire. Tous ces peptides de peau de grenouille contiennent gnralement une grande quantit de rsidus K, il a donc t ncessaire de recourir une raction dactylation afin de lever lambigit Q/K qui existe du fait de la limitation dans la prcision de mesure de masse de linstrumentation MALDI-TOF (20 ppm pour les peptides). Lact! ylation des 4 fractions HPLC nous a permis de lever les ambiguts K/Q et nous a montr que cette raction peut avoir lieu sur les fonctions amine primaires (N-terminal et rsidus K) mais aussi, dans une moindre mesure, sur la fonction amine secondaire des rsidus H. Lactylation complte des peptides conduit la disparition des sites protonables (si aucun rsidu R dans la squence). Ceci entraine la disparition de la molcule monoprotone [M+H]+ au profit des espces [M+Na]+ et [M+K]+. Dans le cas de la prsence dun rsidu P dans la squence de ces peptides, on observe une fragmentation source, prfrentielle en N-terminal du rsidu P. Ces peptides actyls constituent de bons modles pour aborder le phnomne dionisation en MALDI. Link: http://www.chimie.ens.fr/LBM/
68/381
[O22] Use of quantitative proteomics to study function and specificity of an emerging protease family in cell-based assays
Frdric Delolme1, Robin Capomaccio2, Isabelle Zanella-Clon1, Michel Becchi1, David Hulmes2, Christopher Overall3, Catherine Moali2
1 2 3
Institut de Biologie et Chimie des Protines, FR 3302, 7, Passage du Vercors, 69367 Lyon Institut de Biologie et Chimie des Protines, FRE 3310, 7, Passage du Vercors, 69367 Lyon Centre for Blood Research, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3 Vancouver, BC
Contact: Catherine Moali (c.moali@ibcp.fr)
Keywords: Proteins, peptides and small molecules quantification The substrate repertoire of human tolloid-like proteinases (also known as BMP-1, mTLD, mTLL-1 and mTLL-2) has considerably expanded in the past 10 years to include several extracellular matrix components, growth factors and angiogenic factors. These extracellular metalloproteases are now thought to synchronize the biosynthesis of the extracellular matrix with the activation of several growth factors and appear as key players during embryogenesis and tissue repair. However, our current understanding of the physio-pathological roles of BMP1/tolloid-like proteases is limited by the lack of high-throughput approaches to identify novel substrates and/or determine the relevant subset of substrates that are cleaved in specific biological contexts. Quantitative proteomics applied to cell-based assays and tissues is at the moment one of the most powerful techniques to address these questions. Protease-generated fragments, released from the cell-surface or the extracellular matrix, are expected to be found in higher amounts in the supernatants of cells expressing active protease compared to cells expressing inactive protease and this difference can be monitored using iTRAQ labelling of tryptic peptides and quantification with LC-MS/MS. This technique has allowed the identification of several candidate substrates of BMP-1 which have then been studied by western blotting and classical biochemical techniques. However, secreted substrates that do not change cellular compartment after cleavage are missed by this technique and we are now switching to the more exhaustive TAILS technique (Terminal Amine Isotopic Labelling of Substrates) which relies on pre-labelling of whole proteins prior to fragmentation followed by a step of negative selection to enrich the sample in N-terminal peptides (1). Finally, iTRAQ labelling of whole proteins is also used in vitro to identify cleavage sites in complex extracellular substrates bearing glycosaminoglycan chains, as an off-gel alternative to Edman sequencing. (1) Kleifeld, O.; Doucet, A.; auf dem, K. U.; Prudova, A.; Schilling, O.; Kainthan, R. K.; Starr, A. E.; Foster, L. J.; Kizhakkedathu, J. N.; Overall, C. M. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol. 2010, 28 (3), 281-288. Link: www.ibcp.fr
69/381
[O23] Analytical strategy combining NMR, chemical synthesis and mass spectrometry for molecular and structural characterization of plasmapolymerized siloxanes.
Thierry Fouquet1, Jrme Bour1, David Ruch1, Laurence Charles2
1
Advanced Materials and Structures - CRP Henri Tudor, 66, rue de Luxembourg, L-4002 Esch-sur-Alzette (Luxembourg)
2
SACS - Aix-Marseille Universits, Campus de Saint-Jrme - case 512, F-13397 Marseille (France)
Contact: Thierry Fouquet (thierry.fouquet@tudor.lu)
Keywords: Organic and inorganic mass spectrometry Contrary to conventional polymerization techniques, plasma-polymerization at atmospheric pressure leads to thin, poorly soluble and highly cross-linked films, with huge potential for industrial applications. Their microstructure, affected by plasma process parameters and the nature of the precursors, is thus quite difficult to assess. To overcome these difficulties, a complete analytical strategy is proposed combining NMR spectroscopy, organic synthesis and mass spectrometry leading to a detailed molecular and structural characterization of some plasma-polymers. The targeted samples are plasma-polymerized films deposited by means of Atmospheric Pressure plasma generated by Dielectric Barrier Discharge. Two siloxane precursors were plasma-polymerized, either cyclic (octamethylcyclotetrasiloxane, D4) or linear (hexamethyldisiloxane, HMDSO), to synthesize ppD4 and ppHMDSO deposits, respectively. NMR spectroscopy offered a global vision of the deposits as well as an insight in structural features of their insoluble parts by the detection of well-known M, D, T or Q silicon functions for instance. Based on the tandem mass spectrometric behavior of linear methyl-terminated PDMS, molecular and structural information was obtained for the electrosprayed ppD4 soluble part. Distributions of peaks spaced by 282 Da were detected and seen as [OSi(CH3)2]2(OSiCH3)2 cycles bound via either an oxygen atom (A, major species), a OCH2 group (B) or a linear O Si(CH3)2 backbone (C). The expected dimer of the distribution (A) as well as some potential oligomers of the (C) series were then synthesized by conventional chemistry and submitted to CID, so as to compare the MS/MS patterns of plasma and synthesized species and validate the structural elucidation. The ESI-MS spectrum of ppHMDSO revealed at least eight oligomeric distributions, further noted Cn, with n=0-7 indicating the degree of cross-linkage. MS/MS spectra of the oligomers from a given Cn distribution showed n major product ion series. C0 oligomers exhibit MS/MS spectra quite similar to those obtained from linear CH3-PDMS with only one product ion series. In contrast, interpretation of CID data of more cross-linked oligomer adducts required alternative models, amongst commercial T-branched PDMS was shown to be promising for the C1 and C2 series. In addition, several new home-made cyclolinear PDMS would constitute promising models for the highest cross-linked distributions. As a perspective, an aminolysis reaction allowed a entire dissolution of a homemade highly cross-linked silicon rubber, leading to hydroxy-terminated and cyclic siloxanes. Applied to plasma-polymers, it would be an elegant breakthrough for the complete structural characterization of plasma-polymers, allowing the insoluble part to be also mass-analyzed by ESI-MS(/MS).
70/381
[O24] Evaluating the accuracy of the positioning of phosphorylations by MS/MS spectra

Markus Muller1, Erik Ahrne2, Frederic Nikitin1, Frederique Lisacek1
1 2
Proteome Informatics Group, Swiss Institute of Bioinformatics, 1, rue Michel Sevet, 1211 Geneve, Suisse Proteomics Core Facility Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Bale, Suisse
Contact: Markus Muller (markus.mueller@isb-sib.ch)
Keywords: Signaling, interactomics and post-translational modifications, Bioinformatics et biostatistics dedicated to proteomics, Systems biology Many large scale phosphorylation studies ([1][2][3]) have been performed yielding thousands of identified phospho-petides and their positions of phosphorylated amino acids. These studies provide new insights into the paramount importance of phosphorylation as a regulator of biochemical processes. While the statistical false discovery rate (FDR) of the peptide identifications can be controlled by using decoy databases, the error in the assignment of the phosphorylation positions is rarely reported and usually only the position with the highest score is reported. Recently, we introduced QuickMod, a tool to identify modified variants of MS/MS library spectra [4]. We showed that the spectra of unmodified peptides bear considerable similarity to their modified counterparts. Even though the peak intensities can be strongly altered, the fragmentation sites are quite conserved between modified and unmodified peptides. QuickMod implements a spectrum alignment and modification positioning algorithm which uses the information from the unmodified library spectra. It calculates and reports a score for all possible positions of the modifications on the peptide as well as a statistical significance for all these positions. Using publicly available data from large scale phosphorylation studies we extract those phospho-peptides, which have a matching unmodified library spectrum of the same charge and fragmentation technique. We investigate the agreement of the phospho-site positions reported in these data and the results from our spectrum library search for those phospho-peptides. An initial study using a dataset of 1000 doubly and triply charged phosphor-peptide spectra showed that phospho-site assignments are often ambiguous when several serine and threonine residues are present in close proximity. Here, we investigate the accuracy of phospho-sites in more detail and on a larger dataset. We present some simple rules indicating which modification assignments can be trusted and which ones have to be taken with care. [1] B. Bodenmiller et al., PhosphoPep[mdash]a phosphoproteome resource for systems biology research in Drosophila Kc167 cells, Mol Syst Biol, vol. 3, Oct. 2007. [2] S. A. Beausoleil, J. Villen, S. A. Gerber, J. Rush, and S. P. Gygi, A probability-based approach for high-throughput protein phosphorylation analysis and site localization, Nat Biotech, vol. 24, no. 10, pp. 1285-1292, Oct. 2006. [3] J. V. Olsen et al., Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks, Cell, vol. 127, no. 3, pp. 635-648, Nov. 2006. [4] E. Ahrne, F. Nikitn, F. Lisacek, and M. Muller, QuickMod: A Tool for Open Modifcaton Spectrum Library Searches, Journal of Proteome Research, http://dx.doi.org/10.1021/pr200152g
71/381
[O25] Systems analysis of budding yeast fermentation and respiration

Emmanuelle Becker 1, Aurlie Lardenois 2, Yuchen Liu 2, Bertrand Evrard 2, Rgis Lavigne 2, Charles Pineau 2, Michael Primig 2
1 2 3
University of Rennes 1, Campus de Beaulieu, 35042 Rennes, cedex, France IRSET, Campus de Beaulieu, 35042 Rennes, cedex, France Proteomics Core Facility Biogenouest, Campus de Beaulieu, 35042 Rennes, cedex, France
Contact: Rgis Lavigne (regis.lavigne@univ-rennes1.fr)
Keywords: Systems biology Saccharomyces cerevisiae is a model organism for biological processes such as mitotic growth and meiotic development. Recent advances in genome biology have yielded data on yeast genomes, as well as the transcriptome, proteome and interactome. In the present work, we used a molecular systems biology approach by combining different high-throughput datasets, with the aim to gain new insight into growth and meiotic development processes. Duplicate total protein extracts from logarithmically growing diploid cells cultured in rich medium with glucose (YPD, fermentation) or acetate (YPA, respiration) were run onto a high resolution SDS-polyacrylamide gel, each lane was cut into 30 bands which were digested with Trypsin. Samples were then processed by shotgun LC-MS/MS analysis on a LTQ-Orbitrap XL instrument for protein identification. The resulting information was integrated with the output of our own whole-genome expression profiling experiments using tiling microarrays as well as DNA sequencing data and protein network data available via certified public repositories. We have determined the dynamic proteome of diploid yeast cells in two different metabolic states (fermentation of glucose and respiration of acetate). Our label-free method based on a simple protein extraction, and separation step identified essentially all (93%) of the known proteins present in vegetatively growing diploid SK1 budding yeast cells. The vast majority of the proteins were found in both replicates of each medium, underlining our methods robustness. Small numbers of proteins were identified only in YPD or YPA, and several hundred were not detected in any of our experiments. The latter group contained proteins encoded by genes not expressed under the conditions we tested or by loci found to be deleted or mutated in SK1. Our data on the yeast SK1 proteome together with information on DNA and RNA yielded systems-level insight into vegetative growth under two different metabolic conditions and revealed its surprisingly distinct proteome as compared to the reference strain. This work paves the way for large-scale dynamic proteome profiling of complex processes such as meiotic development.
72/381
[O26] Dveloppement Mthodologique pour la Quantification en Imagerie par Spectromtrie de Masse (qMSI)
Grgory Hamm1, David Bonnel1, Raphael Legouffe1, Fabien Pamelard1, Jean-Marie Delbos2, Franois Bouzom2, Catherine Piveteau3, Nicolas Willand3, Benoit Dprez3, Jonathan Stauber1
1 2 3
ImaBiotech, Campus Cit Scientifique, 59655 Villeneuve dAscq, France Technologie Servier, 25 Rue Eugene Vignat , 45000 Orlans, France INSERM U761, Biostructures & Drug Discovery, Universit Lille Nord de France, 59000 Lille, France
Contact: Grgory Hamm (hamm.gregory@imabiotech.com)
Keywords: Imaging mass spectrometry, Proteins, peptides and small molecules quantification Lobtention de donnes quantitatives partir dune exprience dimagerie par spectromtrie de masse (MSI) reste ce jour un challenge pour notre communaut scientifique. Cette quantification require la mise au point dune mthodologie particulire afin dobtenir une meilleur reproductibilit et homognit des mesures (signal) au mme titre quune courbe de calibration prsentant un minimum de variabilit et une trs bonne linarit. Plusieurs mthodes ont t dcrites dans la littrature mais aucune delles na dapplications universelles. Nous proposons ici deux approches la fois diffrentes et complmentaires en vue de la quantification par imagerie MALDI-MS. Nous tenterons par ce fait de rpondre aux principales difficults prendre en compte dans la dmarche de quantification par MSI, savoir, premirement, la haute dpendance du signal dtect la dposition/proprits de la matrice MALDI ainsi qu sa capacit dextraction ; deuximement, le rendement dionisation spcifique des molcules cibles ; et finalement, leffet de suppression ionique du au tissue. Notre mthodologie a t applique dans le cadre de ltude de la distribution de deux mdicaments, le propranolol (Sigma-Aldrich) et le BDM31343, sur un modle animal (souris) par imagerie MALDI-TOFMS (Autoflex Speed, Bruker). Les protocoles exprimentaux et les paramtres instrumentaux ont t adapts chacune des cibles. Les deux approches choisies pour cette tude consistent, pour la premire, en lutilisation dun broyat dun organe dintrt auquel est ajout une gamme de concentration connue dune molcule cible puis reconstitu par conglation et enfin analys en mme temps quun chantillon trait comportant le compos tudi. Cette premire approche est particulirement adapte ltude de la distribution dun compos cible dans un organe particulier, elle ncessite une plus moins longue prparation et un temps de retraitement des donnes minimum. La seconde approche, quant elle, prend en compte leffet de suppression ionique inhrent au tissu analys par imagerie MALDI-MS en utilisant un facteur de normalisation, nomm TEC (Coefficient dExtinction Tissulaire). Ce coefficient est calcul en comparant le signal dune molcule cible sur et en dehors du tissu (support). Il permet de normaliser les donnes obtenues lors de lanalyse dun chantillon trait par un mdicament donn, la molcule cible prcdemment cite. Cette approche a pour principaux atouts, un temps de prparation limit et dtre adapt ltude de plusieurs organes simultanment. Comme inconvnient, un traitement de donns important est ncessaire. Afin de valider nos deux approches, les rsultats issus de notre mthodologie ont t compars des tudes effectues sur les mmes chantillons par dautres techniques analytiques (lautoradiographie et la LC-MS/MS).
73/381
[O27] Using proteomics to identify AICAR cellular targets

Hans C. Hrlimann1, Imad Bouzaidi Cheikhi1, Stphane Claverol2, Marc Bonneu2, Bertrand Daignan-Fornier1, Benot Pinson1
1
Institut de Biochimie et Gntique Cellulaires CNRS/ Universit Victor Segalen Bordeaux 2 , 1, rue Camille Saint Sans, 33077 Bordeaux
2
Ple Protomique, Centre de Gnomique Fonctionnelle Bordeaux, Universit Victor Segalen Bordeaux 2, 146 rue Lo Saignat, 33076 Bordeaux Contact: Hans C. Hrlimann (hurlimann@ibgc.cnrs.fr)
Keywords: Systems biology AICAR monophosphate (5-Aminoimidazole-4-carboxamide ribonucleoside-5 monophosphate) is a metabolic intermediate of the de novo purine synthesis pathway, presenting highly promising metabolic and antiproliferative properties. One of its known activities is the induction of the AMP dependent kinase (AMPK), explaining many, but not all of its effects. The molecular mechanisms underlying the AMPK independent effects are poorly understood up to now. From yeast (S. cerevisiae) it is known that AICAR effects are multifactorial. This was demonstrated with genetic and metabolic analysis. To better tackle the complexity of the AICAR induced phenomena, we now applied another approach. To systematically screen for cellular targets of AICAR, a proteomic approach was undertaken in yeast, using affinity chromatography and subsequent identification of the binding proteins by mass spectrometry. Like this 62 proteins (~1% of the proteome) were found in at least four of five experiments and defined as AICAR binding proteins. To test the specificity of the affinity chromatography, the same experiment was done for succinyl-AICAR (SAICAR). It occurs just one step before AICAR synthesis and is therefore chemically very close, differing only by an additional succinyl group. These experiments resulted in a list of 70 SAICAR binding proteins and strikingly only five are identical to the previously identified AICAR binding proteins. Interestingly the two different groups of binding proteins also belong to distinct functional groups of proteins. Our conclusion was that specificity of the proteomic approach used is acceptable and that a different action of the two molecules analyzed is expected. Detail studies of candidate proteins were thereafter undertaken in yeast, to further validate the approach. We were furthermore interested in the identification of evolutionary conserved binding proteins, with the assumption in mind that this will allow us to reveal processes regulated by AICAR, conserved between species. We therefore proceeded with the identification of AICAR binding proteins in species from bacteria to man and have now a large overview over the most probable targets of this small molecule. As a second approach to identify proteins affected by AICAR, a drug affinity responsive target sensitivity (DARTS) study is on the way now, applying mass spectrometric identification and label free quantification.
74/381
[O28] Dtection sensible et spcifique des spores de Bacillus anthracis dans des matrices environnementales par immunocapture et spectromtrie de masse en mode MRM.
Jrme Chenau1, Franois Fenaille1, Eric Ezan1, Patricia Lamourette1, Natalie Morel1, Pierre Goossens2, Franois Becher1
1 2
CEA, iBiTec-S, Service de Pharmacologie et dImmunoanalyse, CEA de Saclay, 91191 Gif-sur-Yvette
Institut Pasteur, Toxines et Pathognie Bactrienne, URA 2172 CNRS, 25-28 rue du docteur Roux, 75724 Paris Cedex 15 Contact: Jrme Chenau (jerome.chenau@cea.fr)
Keywords: Proteins, peptides and small molecules quantification , High mass and high resolution mass Spectrometry Lanthrax, maladie infectieuse aige, est li la bactrie Bacillus anthracis (Ba). Cette bactrie gnre une structure rsistante appele spore qui constitue la forme de dissmination de la maladie. Le CDC (Centers for Disease Control) assigne Ba comme agent de classe A, le niveau de risque le plus lev. Lutilisation des spores de Ba comme arme biologique a t souligne lors de lenvoi de lettres contamines aux Etats-Unis en 2001, qui causa 22 cas dont 5 dcs. Il est donc indispensable de dtecter la prsence de spores de Ba dans des chantillons environnementaux potentiellement contamins. Une mthode efficace doit tre la fois sensible, spcifique, reproductible et rapide. La protomique et la MS sont des approches pertinentes pour rpondre cette problmatique. La MS a ici t utilise pour deux applications : la dtection sensible des spores de Ba en mode cibl de type MRM et la dcouverte de nouveaux biomarqueurs spcifiques. Nous avons tout dabord dvelopp un procd analytique original de type immuno-LC/MS/MS adapt la dtection des spores de Ba. Il combine la spcificit et la sensibilit de deux techniques complmentaires : immunocapture et MS en mode cibl. Limmunocapture est ralise directement sur les spores intactes. Elle permet de les extraire spcifiquement de matrices environnementales complexes et entraine un gain en sensibilit. La seconde tape consiste dtecter en mode MRM la protine SASP-B (Small Acid-soluble Spore protein B). Cette protine est exprime exclusivement dans les spores et en quantit abondante. Un isoforme particulier a t pralablement dcrit pour les spores de Ba et permet ainsi de les discriminer des autres bactries du groupe cereus phylogntiquement trs proches. La limite de dtection (LOD) de notre approche est de 10^3 CFU/mL dans le lait et 7.10^3 spores dtectes dans 10 mg de terre. Ces LOD sont au niveau de la dose infectieuse minimum (ID50 : 10^3-5.10^4) et proches de celles obtenues par PCR. Cependant le suivi dun seul biomarqueur peut prsenter un biais en terme de spcificit. Nous avons ainsi cherch en parallle identifier de nouveaux marqueurs discriminant Ba des autres bactries du groupe cereus, ceci afin de diversifier les protines cibles et ainsi augmenter encore la spcificit de dtection. Dans ce but, des extraits de spores issus de 38 souches (10 Ba versus 28 B. cereus et B. thuringiensis) ont t analyss par MS haute rsolution sur un LTQOrbitrap. Sept protines prsentant des mutations spcifiques de Ba ont t mises en vidence pour la premire fois. Le suivi de ces marqueurs, en plus de la SASP-B, par MRM multiplex permet de dtecter trs spcifiquement les spores de Ba. En conclusion, ces travaux prsentent une utilisation originale de la MS pour la dtection sensible et spcifique des spores de Ba. Ce procd pourra tre tendu la dtection dautres bactries de la menace.
75/381
[O29] Identification and characterization of new protein partners of serotonin transporter

Pascal Seyer1, Mathieu Seimandi1, Franck Vandermoere1, Jol Bockaert1, Philippe Marin1
1
Institut de Gnomique Fonctionnelle - CNRS UMR5203 - INSERM U661 Universits Montpellier 1 & 2, 141 rue de la Cardonille, 34094 Montpellier, France Contact: Pascal Seyer (pascal.seyer@igf.cnrs.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications The plasma membrane serotonin transporter (SERT) plays a critical role in the regulation of serotonergic transmission by enabling serotonin reuptake into the cells. This transporter is of major pharmacological and clinical interest, particularly as it represents one of the primary targets of several widely prescribed antidepressants. It is now well established that SERT does not function as an isolated protein. SERT functional activity is regulated by a combination of multiple mechanisms including both post-translational modifications and association with intracellular proteins. During the last decade, several SERT-interacting proteins have been identified, principally by means of yeast two-hybrid screens. We have recently used a proteomic approach that enabled us to characterize a reciprocal modulation of SERT and neuronal NO Synthase (nNOS) activity mediated by their physical interaction. To get further insight into SERTassociated protein complex, we used high-resolution mass spectrometry to identify novel proteins interacting with SERT C-terminus, or whole SERT protein expressed in two different cell culture models. This shotgun analysis of SERT interactome led us to identify several new partners of SERT. These include Calcineurin, a calcium-dependent serine/threonine phosphatase, ASCT2 (Alanine Serine Cysteine Transporter 2), a neutral amino acid transporter, VELI-3, a PDZ domain-containing protein which regulates plasma membrane distribution of several receptors and transporters, and a set of proteins related to the SNARE complex, possibly involved in SERT export to the plasma membrane. Moreover, we showed that both physical interaction of SERT with Calcineurin and Calcineurin phosphatase activity increase SERT plasma membrane expression and 5HT uptake via SERT. In addition, co-expression of VELI-3 with SERT likewise increases 5HT uptake, whereas co-expression of ASCT2 decreases SERT activity, possibly via modification of its glycosylation status. Collectively, these proteomic studies identify novel regulation mechanisms of SERT activity that might influence serotonergic transmission.
76/381
[O30] Une suite logicielle pour la protomique interface sur une grille de calcul. Utilisation d'algorithmes libres pour l'identification MS/MS, le squenage de novo et l'annotation fonctionnelle.
Christine Carapito1, Alexandre Burel1, Patrick Guterl1, Jrme Pansanel2, Fabrice Bertile1, Alain Van Dorsselaer1
1
Laboratoire de Spectromtrie de Masse BioOrganique, DSA, IPHC, UMR 7178, CNRS, Universit de Strasbourg, 25, Rue Becquerel, 67087 Strasbourg, France
2
Dpartement Recherches Subatomiques, DRS, IPHC, UMR 7178, CNRS, Universit de Strasbourg, 23, Rue du Loess, 67037 Strasbourg, France Contact: Christine Carapito (ccarapito@unistra.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics Contexte Les progrs instrumentaux en spectromtrie de masse (MS) de ces 20 dernires annes ont conduit au dveloppement dinstruments gnrant des donnes MS/MS de plus en plus volumineuses (du fait dune grande rapidit dacquisition des spectres de fragmentation). Par ailleurs, la soumission des rsultats didentification de protines partir de ces donnes MS/MS est de plus en plus rglemente par les journaux du domaine qui recommandent lutilisation dalgorithmes transparents (open-source) et multiples si possible. Dans ce contexte, afin de rpondre au besoin croissant de puissance de calcul ncessaire linterprtation des donnes MS/MS, une suite logicielle btie sur des logiciels libres a t adapte et amliore sur une grille de calcul. Mthodes et Rsultats La suite logicielle dveloppe lIPHC permet : - de crer, dextraire, de concatner, de formater des banques de squences protiques, notamment partir des banques de squences publiques accessibles telles que NCBInr, UniProtKB, UniProtKB/SwissProt, - de lancer des requtes OMSSA (Open Mass Spectrometry Search Algorithm) pour lidentification de protines partir de donnes MS/MS sur la grille de calcul locale de lIPHC (1024 curs de calculs, site TIER-2 de la grille LHC (Large Hadron Collider)) et mondiale. - dinterprter haut dbit des donnes MS/MS acquises sur des organismes non squencs par squenage de novo suivi de recherches dhomologies de squences. - dextraire de manire automatise les annotations fonctionnelles disponibles sur les protines identifies. Lensemble de ces outils est disponible sur le site https://msda.u-strasbg.fr. Conclusion Ladaptation de la suite logicielle sur une grille de calcul permet de rpondre aux importants besoins de puissance de calcul non accessibles ce jour dans les laboratoires de protomique. En effet, selon le type de requte (nombre de spectres MS/MS, taille de la banque de squences, recherche de modifications post-traductionnelles, squenage de novo, ), un gain dun facteur 100, voire 1000 est obtenu en routine grce la paralllisation des outils et leur lancement sur une grille de calcul. Link: https://msda.u-strasbg.fr
77/381
[O31] Quelle voie pour la caractrisation d isoformes protiques sur coupe de tissu par MALDI in source decay ?
David Calligaris1, Claude Villard1, Therese Schembri1, Daniel Lafitte1
1 2
Proteomique et Innovation Technologique Timone, 27 Bd Jean Moulin, 13385 Marseille CRO2 INSERM U911, 27 bd jean Moulin, 13385 Marseille
Contact: Daniel Lafitte (daniel.lafitte@univmed.fr)
Keywords: Signaling, interactomics and post-translational modifications, Imaging mass spectrometry L'expression des isotypes dune protine est sous le contrle de plusieurs gnes, et les isoformes issus dun mme gne sont produits par pissage alternatif et/ou par modification post-traductionnelle. Ces processus participent la complexit du protome. Ces protines homologues sont souvent fonctionnellement diffrentes et sont impliques dans la rgulation de diffrents processus physiologiques. En spectromtrie de masse, la caractrisation des protines est habituellement effectue en utilisant des approches de type bottom-up par CID ( collision induced dissociation ) ou par PSD ( Post source decay ). Nanmoins, les protocoles associs exigent une digestion pralable des protines, ceci allongeant le temps de traitement des chantillons. Pour caractriser les isotypes dune protine, l'approche top-down par MALDI in-source decay (ISD) est une technique de choix [Brown et al. Anal. Chem. 1995]. Lors de ce processus, une augmentation de la fluence laser conduit la fragmentation des protines [Takayama J. Am. Soc. Mass Spectrom. 2001, Suckau et al. Anal Chem, 2003]. La fragmentation ISD emprunte deux voies, la voie radicalaire produisant majoritairement des ions c et z et la voie thermique produisant des ions b et y. Les travaux mens au sein de notre laboratoire ont indiqu une fragmentation ISD de lextrmit C-terminale de tubuline de cellules HeLa. La tubuline, cible majeure en chimiothrapie anticancreuse, est prsente au sein dun organisme sous diffrents isotypes. A partir dun seul spectre, il a t possible de dterminer la composition isotypique dune solution purifie [Calligaris et al. Anal Chem. 2010]. Pour la caractrisation de biomarqueurs et le dveloppement de nouvelles thrapies, il savre ncessaire de caractriser ces isotypes protiques au sein des tissus. LISD sur coupe de tissu a t rcemment dmontr par le groupe dE. De Pauw [Debois et al. Anal Chem. 2010]. Diverses protines de cristallin de porc ont ainsi t mis en vidence et collocalis par imagerie MALDI coupl lidentification par ISD. Les protines identifies sont majoritairement fragmentes par un processus radicalaire. Notre tude a permis lidentification sur coupe de cerveau de souris disotypes de tubuline. Cette caractrisation par MALDI in source decay est complmentaire des rsultats obtenus par Debois et al. puisque nous dmontrons que certaines protines empruntent plutt la voie thermique que la voie radicalaire pour la fragmentation en source et que cela facilite leur identification dans un mlange complexe. Notre groupe souhait apporter un rel complment lanalyse histopatologique en effet la caractrisation in situ des isotypes de tubuline peut aboutir un meilleur pronostique des tumeurs solides et permettre une avance vers la personnalisation des traitements chimiothrapiques.
Link: http://map.univmed.fr/
78/381
[O32] System biology insights into the global remodeling of proteome and pathogenicity of Bacillus cereus induced by oxydoreduction potential
Grmy Clair1, Catherine Duport3, Jean Armengaud1
1 2 3
Laboratoire de biochimie des systmes perturbs, CEA,DSV,IBEB, pb-17171, 30207 Bagnols-sur-ceze universit d'avignon et des pays du vaucluse, , 84000 avignon UMR A408 SQPOV, domaine saint paul, 84914 avignon
Contact: Grmy Clair (my-g-rem@hotmail.fr)
Keywords: Systems biology Bacillus cereus is a pathogenic agent causing human foodborne disease. Diarrheal syndrome may be the consequence of the production in the hosts small intestine of various extracellular factors, including enterotoxins. In human intestine, pathogens have to deal with a lack of carbohydrates, varying oxygen concentrations and oxydoreduction potential (ORP). While bacterial response to changes in oxygen level has been widely studied, adaptation to various POR has never been documented. We cultured B. cereus in a medium mimicking intestinal conditions under low and high-ORP anoxic conditions (pO2 = 0%) and in full oxic condition (pO2 = 100%). We compared (1) the early secreted and (2) the intracellular protein contents by means of a high-throughput label-free proteomic strategy. A total of 27 biological samples were in-depth analyzed following a system biology approach: record of high-throughput quantitative proteomic data, metabolism modeling, creation of mutant strains, high-throughput proteomics, and so on. After protein extraction and trypsinisation, nano-LC-MS/MS detection of the peptides resulted in a large body of MS/MS spectra acquired (above 1.5 millions). A total of 15,022 unique peptides were assigned to 1,373 proteins. We observed that most proteins identified in secretome are related to pathogenesis. Two-thirds have never been detected before. Changes in secreted protein levels depending on oxygen availability and ORP induce a modification of cytotoxicity towards Caco-2 human epithelial cells (Clair et al, 2010). Detailed study of intracellular proteome associated to metabolic data highlights that a global proteome remodeling occurs in low-ORP anaerobiosis compared to high-ORP anaerobiosis. Candidate proteins possibly involved in this remodeling were identified by analyzing proteomic data. Their major roles were then elucidated by (i) constructing strains lacking these proteins and (ii) analyzing the intracellular-proteome and secretome of the mutant strains (Clair et al, 2011). References : Clair, Roussi, Armengaud, Duport (2010) Mol Cell Proteomics 9:1486-98. Clair, Armengaud, Duport (2011) submitted for publication Link: http://www.mcponline.org/content/9/7/1486.long
79/381
[O33] Identification par trs haute rsolution FT-ICR de processus induits par la charge dans la dcomposition de peptides cycliques en ECD
Carlos Afonso1, Hanane Belghit1, Marie Prot-Taillandier1, Jean-Claude Tabet1
1 2
Institut Parisien de Chimie Molculaire, UMR 7201 CNRS-UPMC, 4 place Jussieu, 75005 Paris, France
Molcules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS-MNHN, 57 rue Cuvier, 75005 Paris, France Contact: Carlos Afonso (carlos.afonso@upmc.fr)
Keywords: High mass and high resolution mass Spectrometry Le squenage des peptides linaires par spectromtrie de masse se fait conventionnellement en mode CID. Cette technique permet la dissociation des ions par apport dnergie vibrationnelle impliquant des fragmentations induites par la charge. Dans le cas de lECD, la dissociation est cause par capture dun lectron lent menant dautres types de dissociations. Cette technique implique des mcanismes qui ne sont pas parfaitement connus notamment pour les peptides cycliques qui ncessitent deux fragmentations conscutives, ce qui rend lattribution trs complexe. Dans la littrature, le modle de la cascade radicalaire est gnralement considr pour expliquer la formation de fragments partir de peptides cycliques[1]. Au cours dun travail prliminaire effectu sur des peptides lasso, il est apparu que la formation dions bj en ECD est due a priori des processus conscutifs induits par la charge [2]. Une premire rupture lie la capture dun lectron ouvre le cycle (clivage c/z) et une deuxime rupture induite par la charge conduit la formation des ions de la srie bj. Au cours de ce travail la dissociation par capture dlectron (ECD) de peptides cycliques tels que la cyclosporine A, a t compare aux dissociations induites par collisions (CID). Cette tude effectue sur un ESI-FT-ICR (Fourier Transform Ion Cyclotron Resonance) a permis, grce la haute rsolution en masse de cet appareil, danalyser finement la composition des diffrents fragments produits par ECD. Nous avons montr que dans le cas de la cyclosporine, en ECD, il existe en plus des fragmentations induites par le radical (cascade radicalaire), une voie de fragmentation comptitive induite par la charge. [1] Leymarie, N.; Costello, C. E.; O'Connor, P. B. Electron capture dissociation initiates a free radical reaction cascade. Journal of the American Chemical Society 2003, 125, 8949-8958. [2] Zirah, S.; Afonso, C.; Linne, U.; Knappe, T. A.; Marahiel, M. A.; Rebuffat, S.; Tabet, J.-C. Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides. J. Am. Soc. Mass Spectrom. 2011, 22, 467-479.
80/381
[O34] Detection de lepo humaine recombinante dans les fluides biologiques equins par lc-ms/ms.
Patrice Garcia1, Ludovic Bailly-Chouriberry1, Maria Lnnberg2, Florence Cormant1, Marie-Agns Popot1, Yves Bonnaire1
1 2
Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, 91370 Verrires le Buisson, France Dept. of Physical and Analytical Chemistry, Uppsala University, PO Box 599, SE-751 24 Uppsala, Sude
Contact: Patrice Garcia (p.garcia@lchfrance.fr)
Keywords: Ionic mobility Lrythropotine humaine recombinante (rHuEPO) est une glycoprotine de 30-34 kDa interdite dutilisation par le Code des Courses. Cependant depuis quelques annes, des cas de dopage avrs ont t observs chez le cheval aux USA et en Europe, mais aussi dernirement chez le dromadaire. De nombreuses mthodes pour diffrencier lEPO quine endogne de lEPO humaine recombinante ont t dveloppes aussi bien en LC-MS/MS quen western-blot (double-blotting, WADA). Toutefois, le temps de dtection est relativement court (48h) et le screening ralis par ELISA reste perfectible. Afin damliorer ce temps de dtection des rHuEPOs dans le plasma et lurine quine, une colonne monolithe greffe avec des anticorps monoclonaux anti-EPO a t value pour la prparation des chantillons. Cette nouvelle technologie, combine avec laffinit de lEPO pour la lectine (MAIIA : Menbrane-Assisted Isoform ImmunoAssay) et analyse par un scanner haute rsolution, a t utilise pour le dpistage de lEPO dans des chantillons plasmatiques et urinaires provenant de chevaux ayant reu des traitements soit dEPREX, soit dARANESP. En parallle, une mthode de purification base exclusivement sur lutilisation de la colonne monolithe anti-EPO et couple la spectromtrie de masse a t dveloppe dans le but de confirmer la prsence des rHuEPOs dans les diffrents milieux tudis, via la dtection de certains peptides caractristiques. Lintroduction de la mobilit ionique au travers du systme FAIMS (high-Field Asymmetric Waveform Ion Mobility Spectrometry, ThermoFisherScientific) au couplage LC-MS/MS a permis daugmenter la slectivit des peptides et ainsi la sensibilit de notre mthode de dtection. Cette nouvelle combinaison analytique permet ainsi de confirmer la prsence de rHuEPOs en un seul jour avec une limite de dtection de lordre de 100 pg/mL. La mthodologie et les rsultats obtenus seront prsents pour illustrer lapport de ce nouveau dveloppement pour le contrle antidopage des chevaux.
81/381
[O35] Lapport de la spectromtrie de masse haute-rsolution lobtention dempreintes molculaires d'chantillons durine. Applications potentielles au dopage.
Agnta Kiss1, Aurlie Fildier1, Marianna Lucio3, Corine Buisson2, Marie-Magdeleine Flament-Waton1, Philippe Schmitt-Kopplin 3, Ccile Cren-Oliv1
1 2
ISA UMR 5280, Service Central d'Analyse (SCA), Chemin du Canal, Echangeur de Solaize, 69390 Solaize
Agence Franaise de Lutte contre le Dopage (AFLD), Dpartement des analyses, 143, avenue Roger Salengro, 92290 Chtenay-Malabry
3
Helmholtz Zentrum Mnchen, German Research Center for Environmental Health , Ingolstaedter Landstrasse 1 , 85764 Neuherberg Contact: Agnta Kiss (a.kiss@sca.cnrs.fr)
Keywords: High mass and high resolution mass Spectrometry L'amplitude et l'volution du dopage demande le dveloppement de techniques de dtection (screening) plus rapides et plus fiables. Une des principales difficults des laboratoires de contrle est l'mergence continue des substances et des mthodes dopantes. Les nouveaux composs prsentent les mmes proprits dopantes, mais leur structure est lgrement modifie et, par consquent, ils ne peuvent pas tre dtects par les moyens classiques. Afin de relever ce dfi, nous avons choisi une approche mtabonomique. Contrairement aux mthodes utilises l'heure actuelle, cette approche n'est pas axe sur la dtection des substances illicites, mais sur leurs effets sur le mtabolisme de l'athlte. Plus prcisment, cette tude vise mettre en vidence des diffrences dordre mtabolique entre diffrents groupes dindividus tels que : des athltes non-dops, des athltes dops, des volontaires, etc. La stratgie choisie comprend cinq tapes, savoir : le choix des chantillons, lobtention des empreintes molculaires, le traitement statistique des donnes, la slection de biomarqueurs potentiels et lidentification de ces derniers. Lurine est une matrice extrmement complexe contenant un nombre impressionnant de mtabolites avec des proprits chimiques et des concentrations trs variables. De ce fait, aprs une tude de faisabilit ralise sur une plateforme LC-Q-TOF, ltude a t oriente vers la spectromtrie de masse de trs haute rsolution FT-ICR. Cette tude a t ralise sur une cohorte de 50 chantillons durine dont 30 chantillons ont t tests positifs (glucocorticodes et bta-2-Agonistes) par lAFLD; et 20 forment le groupe des contrles (athltes non dops et volontaires). Les chantillons ont t analyss dans une mme squence sur un spectromtre de masse FT-ICR en mode positif et ngatif. Les empreintes molculaires ainsi obtenues ont t ensuite traites par analyse multivarie (APC et OPLS-DA) dans le but de classifier les chantillons et mettre en vidence des profils discriminants. La prcision de mesure (< 1 ppm) ainsi que la sensibilit de la technique utilise ont conduit lobtention dempreintes molculaires riches en information et trs qualitatives. Ainsi, plusieurs composs ayant des profils potentiellement discriminants ont t slectionns. Ensuite, en utilisant un gnrateur de formules brutes et la plateforme MassTrix qui regroupe plusieurs bases de donnes une vision globale de la classification des chantillons a t obtenue. La comparaison des signatures urinaires a montr que la mtabonomique pourrait tre un outil complmentaire pour obtenir des informations et des profils riches partir de lurine. De plus, ltape de prparation dchantillon minimale (dilution) et le temps danalyse trs court font delle une technique adapte lanalyse des grandes cohortes dchantillons, notamment dans des tudes cliniques.
82/381
[O36] Nouveaux algorithmes de regroupement pour grer l'identification des protines ou de modifications post-traductionnelles dans les analyses de protomique haut dbit
Benoit Valot1, Olivier Langella1, Ludovic Bonhomme1, Michel Zivy1
1
PAPPSO, UMR de Gntique Vgtale, Ferme du Moulon, 91190 Gif sur Yvette
Contact: Benoit Valot (valot@moulon.inra.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics L'identification des protines par les moteurs de recherche est une tape ncessaire aux analyses de protomique "bottom-up" haut dbit. Elle est base sur l'identification des peptides et sur leur assemblage le long des squences protiques. En gnral les moteurs de recherche se limitent au classement des protines identifies par ordre de score ou de probabilit. Lorsque plusieurs protines contiennent des peptides identiques, un posttraitement est ncessaire pour lever les ambiguts. Classiquement il est bas sur l'limination par regroupement des protines identifies uniquement par des peptides communs d'autres protines. Mais cela ne suffit pas pour grer les cas d'intersections entre groupes de peptides communs. Nous prsentons ici un algorithme original bas sur un deuxime niveau de regroupement, qui runit les protines contenant au moins un peptide en commun. Il permet de dtecter des protines redondantes non limines par le premier regroupement, de connatre prcisment le nombre de peptides communs et spcifiques chaque protine et d'estimer le nombre d'isoformes identifies dans l'chantillon. Par ailleurs nous avons vrifi qu' de rares exceptions prs, ce niveau de classement regroupe les protines par fonction. Nous prsenterons un cas extrme d'utilisation avec une analyse de mtaprotomique bactrienne. Les recherches systmatiques de modifications post-traductionnelles conduisent souvent une incertitude de position quand plusieurs rsidus susceptibles d'tre modifis sont proches dans la squence. En gnral, les moteurs de recherche proposent les positions les plus probables en analysant sparment chaque spectre, ce qui conduit parfois des rsultats contradictoires pour un mme site suivant le spectre analys. Nous prsentons ici un algorithme innovant qui regroupe l'ensemble des spectres MS/MS identifiant une modification sur des rsidus voisins, de manire tenir compte de l'ensemble des spectres simultanment pour lever l'incertitude de position. Ceci permet de dnombrer correctement les sites modifis et les protines qui les contiennent. Nous prsenterons un exemple d'utilisation sur une analyse du phosphoprotome du mas. Ces deux algorithmes ont t implments dans le logiciel X!Tandem Pipeline utilisant le moteur de recherche X!tandem. Il est disponible gratuitement http://pappso.inra.fr/bioinfo/xtandempipeline/ et distribu sous licence GPL. Link: http://pappso.inra.fr/
83/381
[O37] Post Translational Modifications and Pathogenesis of Bacterial Meningitis Is a one shot Approach for Complete Mapping Realistic?
Joseph Gault1, Christian Malosse1, Jean-Michel Camadro2, Guillaume Dumnil3, Julia Chamot-Rooke1
1 2 3
CNRS UMR 7651 DCMR, cole Polytechnique, 91128 Palaiseau, France CNRS UMR 7592, Institut Jacques Monod, Universit Paris-Diderot, 75013 Paris, France INSERM U970, Paris Cardiovascular Research Center, 75015 Paris, France
Contact: Joseph Gault (gault@dcmr.polytechnique.fr)
Keywords: Systems biology Post translational modifications (PTMs) are increasingly being revealed as key intermediates in pathogenesis pathways1-3. Understanding their role on the molecular level not only greatly increases our comprehension of disease mechanisms but provides an essential basis onto which human intervention strategies can eventually be built. Detection and localisation of PTM is not an easy task and mass spectrometry, which is a fast and sensitive analytical tool, is often implicated somewhere in the process. In recent work we identified an important PTM on the type IV pili of Neisseria meningitidis. Pili are extracellular, filamentous organelles implicated in a variety of life processes including bacterial aggregation and host cell attachment. The pilus itself is a macro polymer built up of the repeating 17.5kDa protein unit pilE, which is arranged helically to create long and flexible fibres. The glycerophosphate modification, present on serine 93 of this protein, is induced in vivo after several hours of host cell contact. We hypothesise that subsequent alteration of the pilus surface, once modified by the phosphoester, ultimately leads to the dissemination of the bacterium; a step that forcibly precedes invasive infection4. In this work a combination of top-down and bottom-up methods were used for structural characterisation of pilE and to localise the modifications of interest. However, an effective topdown approach for localisation of all PTMs had at that time not been developed. Here we present top-down results using both Orbitrap and FT-ICR mass spectrometers and a variety of fragmentation methods, ECD, ETD, CAD, HCAD, IRPMD and combinations thereof, for the localisation of the 5 modifications present on this protein. Unusually we also present comprehensive profiling of several of these fragmentation techniques performed on the pilE protein itself. Aspects of the techniques, fragmentation mechanisms and applicability to other systems will be discussed along with the major implications of a one shot approach to understanding bacterial dissemination. 1S. Subramaniam et al., Science, 324, 1327-1330 (2009) 2A. Oueslati et al., Progress in Brain Research, 183, 115-145 (2010) 3J.K. Kimet al., Molecular & Cellular Proteomics, 2, 7, 453-62 (2003) 4J.Chamot-Rooke et al., Science, 331, 778-782 (2011)
84/381
[O38] Bladder Cancer Biomarker Candidates Discovery in a Multi-site Patients Cohort: a Label-free Quantitative Proteomics Study.
Magali Court1, Mourad Mellal1, Madalen Le Gorrec1, Yves Allory2, Nria Malats3, Christophe Bruley1, Elodie Duriez4, Bruno Domon4, Jrme Garin1, Christophe D. Masselon1
1
Laboratoire d'tude la Dynamique des Protome, BGE, iRTSV, 17 rue des Martyrs, 38054 Grenoble Cedex France
2 3 4
APHP Hpital Henri Mondor, , Crteil - France CNIO, , Madrid - Spain CRP Sant , , Luxembourg - Luxembourg
Contact: Christophe D. Masselon (Christophe.Masselon@cea.fr)
Keywords: Clinical proteomics, Proteins, peptides and small molecules quantification Protein biomarkers discovery of renal or urinary tract diseases in human urine has recently garnered tremendous interest. This fluid represents an ideal diagnostic analyte for bladder cancer, since the tumor is literally bathed in urine. The EU-FP7 funded DECanBio project aims at implementing a generic strategy for protein biomarker discovery and validation. Using state of the art MS instrumentation for quantitative proteins analysis, the primary objective is to provide a restricted number of urinary biomarkers; ultimately enabling the detection of bladder tumour recurrences. As a first step toward this goal, a label-free quantitative proteomics approach was used to compare urine protein profiles among a multi-site cohort consisting of 99 patients including various grades of bladder cancer, and age and sex matched controls. A label-free quantitative proteomics approach based on the Accurate Mass and Time (AMT) tag strategy has been pursued. This strategy allows the analysis of the many samples (tens to hundreds) required at the candidate discovery phase in a reasonable time-frame (a few weeks) while providing a broad proteome coverage and a suitable dynamic range of peptides quantification. A multi-site cohort (France and Spain) was collected according to guidelines developed within the context of the DECanBio project. Proteins were extracted using organic solvent precipitation and digested in-solution as described earlier (Court et al. Proteomics, in press). Samples were analyzed in triplicate and in random order on a Ultimate 3000 nano-HPLC system (Dionex) coupled to an LTQ-Orbitrap XL(Thermo) mass spectrometer. An AMT tag database was generated from >1200 LC-MS/MS analyses of bladder cancer incident, recurrent and control cases. This database constitutes an in-depth repository of all peptides previously detected in urine by our group and their corresponding proteins. It contains over 18,000 non-redundant peptide entries, representing more than 2,000 urinary proteins. In semiquantitative LC-MS analyses, over 1,200 proteins were monitored and quantified over the discovery cohort. The peptides detected spanned a relative abundance range of more than 4 orders of magnitude. To extract lists of differentially abundant proteins between the cohorts sub-populations, two different statistical analysis methods were applied: a modified version of the so-called Spectral Index (SpI) approach and a mixed effects ANOVA. The comparison of healthy versus cancer samples by the two methods resulted in a restricted list of proteins common to both approaches. These proteins are being reviewed using bioinformatics approaches to determine the final list of candidates, which will be evaluated on a different cohort during the subsequent phase of the DECanBio project using absolute quantification by LC-SRM.
85/381
86/381
Posters
87/381
[P1] Effects of metallic nanoparticles on murine macrophages

Sarah Triboulet1, Catherine Aude-Garcia1, Vronique Collin-Faure1, Dvy Diallo1, Hlne Diemer2, Alain Van Dorsselaer2, Thierry Rabilloud1
1
CEA/DSV/iRTSV, Laboratoire de Chimie et Biologie des Mtaux, UMR CEA -Universit Joseph Fourier-CNRS 5249, 17 rue des Martyrs , 38000 Grenoble, France
2
IPHC, Institut Pluridisciplinaire Hubert Curien, UMR CNRS- Universit de Strasbourg 7178, 23 rue du Loess, 67000 Strasbourg, France Contact: Sarah Triboulet (sarah.triboulet@gmail.com)
Keywords: Systems biology Metallic nanoparticles are more and more widely used for consumer products, such as cosmetics, as well as industrial processes. However, so far, few data are available on their potential impact on health and environment. To improve our knowledge and better assess the risks of toxicity, it is necessary to study cellular responses to these particles. As one of the most active scavenger cell type, macrophages appear as a pertinent model to evaluate nanoparticles toxicity. The aim of this study was to analyse the effects of several metallic nanoparticles (Cu, CuO, and ZnO) on the proteome of J774A.1 and RAW264.7 murine macrophage cell lines. The effects were compared to that of the corresponding ions when available (Cu2+, Zn2+). Proteomic analyses were conducted by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. Targeted assays and functional analysis tests, such as glutathione levels, inflammatory mediators and phagocytosis assays, were also performed to confirm and validate the data. Our results showed that Cu and CuO nanoparticles show toxicity at rather low dosages (5-15 g/ml) and inhibit phagocytosis. Analyses of total proteome changes showed changes in proteins involved in oxidative stress (glutamatecysteine-ligase, ferritin) signal transduction, and translation machinery. Our results demonstrated that the chemical nature of the particles was the essential factor, but that within the same family, the size and shapes were also important parameters. Although more research needs to be done, these data are essential to a better and safer use of the concerned nanoparticles. Link: http://www-dsv.cea.fr/dsv/instituts/institut-de-recherches-en-technologies-et-sciencespour-le-vivant-irtsv
88/381
[P2] Apport dune nouvelle technique de dtection et de quantification des anticorps no-synthtiss par le nouveau-n
Clia Dechavanne1, Franois Guillonneau2, Lala Sago2, Virginie Salnot2, Evelyne Guitard3, Philippe Chafey4, Marie-Paule Lefranc5, Jean-Michel Dugoujon3, Patrick Mayeux2, Florence Migot-Nabias1
1
Institut de Recherche pour le Dveloppement, UMR 216 Mre et enfant face aux infections tropicales, Facult de Pharmacie, Universit Paris Descartes, 4 avenue de lObservatoire, 75006 Paris, France
2 3
Plate-forme protomique de lUniversit Paris Descartes, 22 rue Mchain, 75014 Paris, France
Laboratoire d'Anthropologie Molculaire et Imagerie de Synthse, UMR 5288, Universit Paul Sabatier Toulouse III, 37 alles Jules-Guesde, 31073 Toulouse, France
4 5
Institut Cochin, Inserm U1016, 22 rue Mchain, 75014 Paris, France
Laboratoire dImmunoGntique Molculaire, Universit Montpellier 2, UPR CNRS 1142, Institut de Gntique Humaine, 141, rue de la Cardonille , 34396 Montpellier Cedex 5, France Contact: Clia Dechavanne (celia.dechavanne@ird.fr)
Keywords: Proteins, peptides and small molecules quantification , Clinical proteomics Cette tude permet pour la premire fois de dtecter et de mesurer les anticorps nosynthtiss par le nouveau-n en les distinguant des anticorps maternels transmis in utero. La mthode propose utilise des caractristiques individuelles des immunoglobulines G3 (IgG3) lies au polymorphisme des allotypes G3m situs sur les domaines constants des chanes lourdes. Plusieurs peptides prototypiques ont t identifis. Des expriences prliminaires ont t effectues sur du plasma d'adulte dont les allotypes G3m ont t dtermins par la mthode qualitative de rfrence dimmuno-hmatologie. Les IgG3 totales ont t purifies par chromatographie d'affinit puis digres en gel ou en solution par une combinaison de protases. Les peptides obtenus ont t dtects par spectromtrie de masse (Maldi TOF-TOF, Orbitrap). La sensibilit de la mthode a t vrifie laide de ratios volumiques dchantillons plasmatiques de 2 adultes homozygotes pour des polymorphismes dallotypes distincts. Une approche label free a fourni une information semi-quantitative qui sera applique sur des IgG3 totales purifies partir dchantillons plasmatiques d'une mre et de son enfant prlev trimestriellement de la naissance 9 mois. La quantification ultrieure des IgG spcifiques de nouveau-ns apportera des connaissances importantes sur l'acquisition de limmunit et sera un prcieux outil de diagnostic indirect pour certaines maladies transmission verticale.
89/381
[P3] Dialogue molculaire dans la symbiose mycorhizienne

Virginie Puech Pags1, Vrna Poinsot3, Jean Denari2, Jean Charles Portais4, Guillaume Bcard1
1 2 3 4
LRSV, 24 Ch Borde Rouge, 31326 Castanet Tolosan LIPM, 24 Ch Borde Rouge, 31326 Castanet Tolosan IMRCP, 118 r de Nabonne, 31062 Toulouse ISBP, INSA, 31400 Toulouse
Contact: Virginie Puech Pags (puech@lrsv.ups-tlse.fr)
Keywords: Signaling, interactomics and post-translational modifications Vivant en symbiose avec une grande majorit des plantes terrestres, les champignons mycorhiziens arbuscules (MA) constituent un enjeu pour les applications en agriculture durable. Lenjeu agronomique repose sur lexploitation de leur aptitude symbiotique transfrer de leau et des sels minraux la plante partenaire, amliorer les dfenses de la plante contre les pathognes, pour finalement limiter les besoins dirrigation et dintrants chimiques dans les productions vgtales (engrais et pesticides). Depuis plusieurs annes, lquipe de G. Bcard sintresse aux mcanismes de reconnaissance des deux partenaires dans ltape pr-symbiotique. Dans un premier temps, nous avons ralis la caractrisation des molcules vgtales permettant lactivation du champignon mycorhizien, les strigolactones (Besserer et al, PLoS Biol, 2006, Gomez et al, Nature, 2008), via une collaboration avec lquipe de JC Portais (INSA). Dans un second temps, nous avons caractris les molcules fongiques activant la plante, les facteurs Mycs LCOs (Maillet el al, Nature, 2011), via une collaboration avec les quipes de J Dnari (LIPM) et V. Poinsot (IMRCP). Ces molcules ont t caractrises et quantifies par spectromtrie de masse, LC-Q-TOF (ITAV) et LC-Q TRAP (plateforme Metatoul). Nous essayons maintenant de comprendre leur production et rgulation et de chercher dautres molcules actives.
90/381
[P4] Caractrisation des RCPGs par spectromtrie de masse : les apports dune approche multi-enzymatique
Noelle POTIER1, Lauriane KUHN3, Mlanie LEBRETON1, Celine CUNY2, Olivier BORNERT2, Fatima ALKHALFIOUI2, Emmanuelle LEIZE1, Renaud WAGNER2
1
Laboratoire de Dynamique et Structure Molculaire par spectromtrie de masse, Institut de Chimie de Strasbourg, 1 rue B. Pascal, 67008 Strasbourg
2 3
Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, boulevard Sebastien Brant, 67412 Illkirch Plateforme Protomique de l'Esplanade, IBMC, 15 rue Descartes, 67084 Strasbourg
Contact: Noelle POTIER (npotier@unistra.fr)
Keywords: Signaling, interactomics and post-translational modifications Les stratgies danalyse protomique classiquement utilises pour ltude de Protines Membranaires (PM) ( shotgun ou gel 1D) conduisent souvent lidentification de protines cibles, mais le faible taux de recouvrement de squence li au faible nombre de peptides gnrs ne permet pas une caractrisation complte. A laide dun systme modle, les Rcepteurs Coupls aux Protines G (RCPG) produits et purifis partir dun systme levure recombinant, nous avons dvelopp un protocole visant lobtention dun recouvrement de squence maximal, ncessaire la mise en vidence de modifications post-traductionnelles ou disoformes. Ce protocole, appliqu sur 5 RCPGs, est bas sur la fusion des rsultats obtenus par laction de trois agents de digestion sur une mme bande de gel 1D : un agent de digestion spcifique (trypsine), un agent de digestion peu spcifique (chymotrypsine), un agent de digestion chimique (CNBr). Alors quils partagent une mme structure 7 domaines transmembranaires, les taux de recouvrement de squence obtenus sur nos 5 RCPGs aprs une digestion in-gel la trypsine se sont avrs trs variables en fonction du rcepteur considr et cela quelles que soient les conditions dextraction des peptides (prsence de dtergent, concentration leve en acide). En revanche, lutilisation de la chymotrypsine et des doubles digestions CNBr/trypsine sest rvle trs utile en permettant (i) de valider lexactitude de certains domaines grce la dtection de peptides dont les squences se chevauchent partiellement, (ii) daugmenter significativement le taux de recouvrement de squence grce lhydrolyse de plusieurs domaines transmembranaires et (iii) didentifier des domaines possdant des modifications posttraductionnelles. Des taux de recouvrement de squence pouvant aller jusqu 80% dans le cas du rcepteur Purinergic P2RY1 nous ont permis de mettre en vidence la prsence dune Nglycosylation sur lextrmit N-terminale. Laction dendoglycosidases, suivie par MALDI, a montr que les deux asparagines N11 et N27 sont modifies par une structure de type high mannose . Dans tous les cas, la complmentarit des masses mesures par MALDI et par nanoLC-MS/MS a t exploite. Link: http://www-chimie.u-strasbg.fr/ldsm2
91/381
[P5] Characterization of the interaction of frataxin with the de novo iron sulfur biosynthesis complex ISCU/NFS1/ISD11
Adeline PAGE1, Stphane Schmucker2, Alain Martelli2, Florent Colin2, Marie Wattenhofer-Donz2, Laurence Reutenauer2, Hlne Puccio2
1 2
Proteomic Platform, IGBMC, 1, rue laurent Fries, 67404 Illkirch, France
Department of Translational Medecine and Neurogenetics; Chaire de gntique Humaine, Collge de France, 1, rue laurent Fries, 67404 Illkirch, France
3
IGBMC, UMR CNRS 7104, Inserm U596, University of Strasbourg, 1, rue laurent Fries, 67404 Illkirch, France
Contact: Adeline PAGE (adeline.page@igbmc.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications Human Frataxin, the mitochondrial protein deficient in Friedreich ataxia (FRDA), a rare autosomal recessive neurodegenerative disease associated with hypertophic cardiomyopthy, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis. In this study, we show data providing new insights into the interaction of mammalian frataxin with component of the Fe-S cluster assembly machinery. Different mass spectrometry analyses were realized to determine first the interacting partners of human frataxin and then the molecular weight of the frataxin-containing complexes. The interacting partners of human frataxin were identified by coupling immunoprecipitation (IP) with nanoLC-nanoESI-MS/MS analysis. IP was performed from HeLa mitochondrial extracts expressing a recombinant human frataxin with C-terminal flag epitope (hFXN-Flag). Three common proteins (ISCU, NFS1 and ISD11) were identified by mass spectrometry from two independent experiments to specifically co-immunoprecipited with hFXN-Flag. These data indicate that the main endogenous interactors of frataxin are ISCU, NFS1 and ISD11, three components of the core Fe-S assembly complex. By co-expression of murine proteins in bacteria, we were able to isolate a quaternary complex containing mFXN, mISCU, mNFS1 and mISD11. The molecular weight of the quaternary complex was determined by native electrospray mass spectrometry analysis. For that, two complexes, GST-mFXN/mISCU/HIS-mNFS1/mISD11 and mFXN-HIS/mISCU/mNFS1/mISD11, were purified by gel filtration and analysed by MS. Molecular weights of 237 kDa and 190 kDa were deduced for the GST-mFXN/mISCU/HIS-mNFS1/mISD11 and mFXN-HIS/mISCU/mNFS1/mISD11 complexes respectively. These results suggest that the interaction of frataxin with the ISCU/NFS1/ISD11 complex most likely defines a function of frataxin in the early steps of de novo Fe-S cluster biosynthesis and provide new elements, important for further understanding this crucial biological process. The perspectives of this work are to obtain the 3D-structure of the quaternary complex by X-ray crystallography and to determine the exact function of frataxin in the complex. Supplemental mass spectrometry analysis will allow us to analyse the different biochemical states of the de novo biosynthesis complexes (presence of iron and sulfur) occurring upon frataxin association with the ISCU/NFS1/ISD11 complex. In addition, interactors identification by IP coupled to mass spectrometry and biochemical characterization of isolated complexes will be performed on other components of the Fe-S cluster biosynthesis machinery to better understand molecular events involved in Fe-S biogenesis.
Link: http://www.igbmc.fr/
92/381
[P6] ITRAQ: a method to elucidate cellular responses to Zearalenone mycotoxin.

Amel Chatti Gazzah1, Luc Camoin2, Salwa Abid1, Moncef Ladjimi3, Hassen Bacha1
1
Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Rue Avicenne, 5000 Monastir , Tunisia
2 3
Inserm,U891, CRCM, Marseille Protomique, , , F-13009 Marseille, France
Laboratory of Genetic and Cellular Biology, CNRS, UMR 8159, Versailles St-Quentin University, 45 Avenue des Etats-Unis ,, 78045 Versailles 78035, France. Contact: Amel Chatti Gazzah (amelbabbouimmuno@yahoo.fr)
Keywords: Proteins, peptides and small molecules quantification Zearalenone (ZEN) is a secondary metabolite produced by some Fusarium species that contaminate a large variety of grains and feedstuffs worldwide [1]. ZEN has been associated with a wide variety of adverse health effects including be hepatotoxic, hematologic, immunotoxic and genotoxic. In order to better understood the mechanism of ZEN toxicity. A proteomic approach was applied to characterize cellular responses of hepatocarcinoma cells (HepG2) to ZEN exposure. Protein extracts from cultured HepG2 cells treated with 100M ZEN for 8 hours, as well as extracts from control cells. The screening method applied to compare proteome was based on the stable isotope approach isobaric tagging for relative and absolute quantification (iTRAQ). This study identified about 982 proteins among which identification and quantification of peptides and their corresponding proteins were accomplished by MALDI-TOF mass spectrometry (MS). Ingenuity Pathways Analysis (IPA) software was then used to determine the biological functions and canonical pathways associated with the ZEN-responsive proteins.
93/381
[P7] ANALYSE PAR IMAGERIES PAR SPECTROMETRIE DE MASSE TOF-SIMS et MALDITOF DE LEFFET DU BUTYRATE DARGININE SUR LA COMPOSITION LIPIDIQUE DU MUSCLE CARDIAQUE DE SOURIS MODELES DE LA MYOPATHIE DE DUCHENNE
Bryl DONFACK1, Sara VIANELLO2, Claudia BICH1, David TOUBOUL1, Sabine DE LA PORTE2, Alain BRUNELLE1
1
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
2
Centre de Recherche de Gif, Institut de Neurobiologie Alfred Fessard, CNRS, Avenue de la Terrasse, 91198 Gifsur-Yvette Cedex, France Contact: Claudia BICH (claudia.bich@icsn.cnrs-gif.fr)
Keywords: Imaging mass spectrometry La myopathie de Duchenne est une maladie gonosomale caractrise par une dgnrescence des tissus musculaires. Latteinte du muscle cardiaque reprsente la premire cause de mortalit chez les malades.Le butyrate darginine semble protger le muscle cardiaque de cette dgnrescence lorsquil est administr vers lge de 6 semaines (dbut de latteinte cardiaque) chez les souris modles mdx. Des colorations histologiques de curs de souris saines, de souris mdx ayant reues une solution saline (mdx saline) et de souris mdx traites par du butyrate darginine (mdx BA) montrent que les curs de ces dernires prsentent des zones dstructures moins importantes que les curs de souris mdx saline.Des tudes prcdentesralises sur du muscle squelettique de souris avaient montr que le rapport (R1) entre les phospholipides PC34 :2 et PC34 :1 est suprieur 1 dans le muscle sain (zones structures) alors quil est infrieur 1 dans les zones dstructures des souris mdx. Cette inversion a t caractrise par imagerie par spectromtrie de masse dions secondaires (TOF-SIMS) et par spectromtrie de masse de dsorption/ionisation laser assiste par matrice (MALDI-MS) qui, couples un analyseur par temps de vol (TOF), sont des techniques de choix pour lanalyse en surface des lipides dans les tissus biologiques. Ces deux techniques ont t utilisespour vrifierleffet bnfique du butyrate darginine sur les souris mdx et dterminer et localiser les lipides membranairesdans les curs de souris. Un traitement de BA (800 mg/kg/j), dune dure de 7 semaines a t administr par voie intrapritonale des souris ges de 6 semaines (prsence de lsions lgres dans le cur).14 semaines, les souris ont t sacrifies, les curs ont t prlevs etcongels. Des coupes conscutives dpaisseur 16m ont t ralises -20C laide dun cryostat. Lesimages ioniques ont t obtenues grce aux spectromtresTOF-SIMS (analyse directe de la coupe sans dpt de matrice) et MALDI-TOF (aprs dpt de matrice 9-aminoacridine pour le mode ngatif et dacide -cyano-4-hydroxycinnamique pour le mode positif). La variation du rapport R1 entre les phospholipides PC34:2 et PC34:1 a t tudiedans le cur, un tissu qui ne rgnre pas. Les rsultats ont t compars avec ceux obtenus dans le muscle squelettique. Des variations contraires celles obtenues dans ce dernier ont t observes : dans les curs sains (tissus structur), R1<1 et dans les curs mdx saline et BA (tissus dstructur), R1>1. De plus,limagerie par spectromtrie de masse nous a permisdidentifierin situ lesions de lipides m/z 772.6,m/z798.6,et le cholestrol, quisemblent tre caractristiques des zones dstructures du cur (souris mdx saline et BA). Ces rsultats montrent que limagerie par spectromtrie de masse est un outil important pour ltude in situ des variations lipidiques associs une maladie dgnrative.
94/381
[P8] MALDI vs RSLC-ORBITRAP : Amlioration de lidentification de protines de faible masse molculaire issues de gels 2D
Cdric BROUSSARD1, Bastien LAUMAIN1, Virginie SALNOT1, Franois GUILLONNEAU1, Marjorie LEDUC1, Patrick MAYEUX1, Philippe CHAFEY2, Morgane LE GALL2, Guilhem CLARY2
1 2
3P5 (Plate-forme Protomique de l'Universit Paris-Descartes), 22 rue Mchain, 75014 PARIS E2D (Plate-forme d'Electrophorse Bidimensionnelle de l'Institut Cochin), 22 rue Mchain, 75014 PARIS
Contact: Cdric BROUSSARD (cedric.broussard@inserm.fr)
Keywords: Instrumentation, Separative analysis methods, High mass and high resolution mass Spectrometry Une des activits principales de la plate-forme Protomique 3P5 est lidentification par MALDIMS/MS de digests trypsiques de protines issues de gels 2D. De manire rcurrente, le rendement didentification des protines par cette technique chute considrablement pour des protines au-dessous de 20kDa. Nous avons donc choisi danalyser ces digests la fois par MALDI MS/MS et LC-ESI-MS/MS pour orienter cette catgorie de protines vers une autre mthode analytique. Les protines infrieures 20kDa issues de gels 2D et colores au bleu de Coomassie collodal ont t digres par la trypsine laide dun automate. Les peptides extraits ont t ensuite analyss paralllement sur MALDI-TOF-TOF et sur un systme RSLC/LTQ-Orbitrap-Velos avec un programme gradient rapide permis par la RSLC. En effet, lemploi de la RSLC avec une colonne C18 de faible granulomtrie (taille des particules 2m avec un diamtre des pores de 100) permet par comparaison avec une nano-HPLC classique (3m/100) de travailler des dbits et des pressions suprieurs tout en amliorant la rsolution chromatographique et donc lintensit du signal. Les interrogations effectues sur le serveur local Mascot de 3P5 rvlent une augmentation du rendement moyen des identifications de 20% par le MALDI plus de 90% par lOrbitrap avec dans plus dun cas sur deux, plusieurs protines identifies par spot. Une analyse RSLC-MALDI MS/MS est en cours de dveloppement afin de permettre une comparaison plus quilibre entre les deux spectromtres.
95/381
[P9] Identification de protines de radiosensibilit dans les lymphocytes T par LCMS/MS : application au cancer du sein
Jrme Lacombe2, Alain Mang1, Jrme Solassol1, Andr Plegrin2, David Azria2
1
Laboratoire de biostatistique, d'pidmiologie et de recherche clinique, IURC, EA2415, Universit Montpellier 1, 641 avenue du doyen Giraud, 34093 Montpellier, France
2
Laboratoire d'immunociblage et radiobiologie en oncologie, IRCM, INSERM, U896, 208 rue des apothicaires, 34298 Montpellier, France Contact: Jrme Lacombe (jerome.lacombe@etu.univ-montp1.fr)
Keywords: Clinical proteomics, Proteins, peptides and small molecules quantification Lefficacit du traitement radiothrapeutique en cancrologie est principalement dtermine par la dose totale dlivre sur le foyer tumoral. Cette dose est limite par la tolrance des tissus sains associs au volume dirradiation, laquelle peut provoquer des effets secondaires chez certains patients. En raison de son caractre irrversible et de son impact sur la qualit de vie, cette toxicit tardive est particulirement surveille. Plusieurs tests ont t dvelopps afin de prdire dventuels effets tardifs et ceci afin dadapter le traitement chaque patient. Parmi ces tests, Ozsahin M. et al. ont dmontr que le taux dapoptose radio-induite (TALRI) des lymphocytes T CD8 est inversement corrl au dveloppement de complications radio-induites tardives de grade lev. Toutefois, ce test souffre encore dun nombre trop lev de faux positifs pour tre utilis en clinique. Lobjectif de notre travail est didentifier et de caractriser un profil protique spcifique pouvant tre associ lapparition dune toxicit tardive chez des patients irradis. Pour cela, nous avons analys par une approche de protomique diffrentielle la rponse radio-induite de populations lymphocytaires (TCD4 et TCD8) issues de patientes ayant un TALRI bas et prsentant ou non une toxicit tardive. Nous avons identifi un panel de 189 protines diffrentiellement exprimes entre ces deux populations et susceptibles dtre impliques dans diffrentes voies cellulaires associes la toxicit radio-induite. La validation de ces marqueurs potentiels est en cours de ralisation.
96/381
[P10] Proteomic analysis of the 5-HT6 receptor complex: towards the identification of signaling pathways underlying its physiological functions.
Martial Sveno1, Julie Meffre2, Sverine Chaumont-Dubel2, Clotilde Mannoury La Cour3, Florence Loiseau 3, Jol Bockaert 2, Mark J Millan3, Philippe Marin2
1 2 3
Plate-forme de Protomique Fonctionnelle (FPP), IGF, 141 rue de la cardonille, 34094 Montpellier Institut de Gnomique Fonctionnelle (IGF), CNRS, INSERM, UMI-II, 141 rue de la cardonille, 34094 Montpellier Institut de Recherche Servier, 125 Chemin De Ronde, 78290 Croissy-Sur-Seine
Contact: Martial Sveno (martial.seveno@igf.cnrs.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications The serotonin 6 (5-HT6) receptor is almost exclusively localized in the CNS, predominantly in regions involved in the regulation of cognitive processes such as the cerebral cortex, the hippocampus and the striatum. Correspondingly, it has become an increasingly promising target for the treatment of cognitive deficits associated with various CNS disorders such as schizophrenia. 5-HT6 receptors also participate in neuro-developmental processes, controlling the migration and normal positioning of cortical interneurons. Although 5-HT6 receptors are known to signal through Gs-adenylyl cyclase, coupling pathways and mechanisms controlling their activity remain poorly explored. To address these issues, we have recently used proteomic approaches associating co-immunoprecipitation or affinity chromatography experiments and high-resolution mass spectrometry, to identify novel signalling proteins associated with the receptor. Affinity-purified proteins were separated by SDS-PAGE and systematically digested by trypsin. Generated peptides were analyzed by NanoLC-MS/MS with a Fourier transform tandem mass spectrometer (LTQ Orbitrap XL). Protein identification was performed by using the Mascot algorithm. Management and validation of mass spectrometry data, allowing discrimination of specific receptor partners, were carried out using the web server myProMS v2.3 (Proteome Analysis using Mass Spectrometry, Institut Curie). These proteomic studies revealed interaction of the receptor with 28 specific partners of which several members of the mammalian target of rapamycin (mTOR) cascade. These include mTOR itself and Neurofibromin (Nf1), which are known to control synaptic plasticity, learning and memory formation. They also demonstrated association of the receptor with a network of proteins involved in the actin cytoskeleton dynamics, which is crucial for neurite growth, dendritic spine formation and neuronal migration. Functional studies demonstrated that stimulation of 5-HT6 receptor activated the mTOR pathway in various brain areas including the prefrontal cortex, the site of modulation of social memory by 5-HT6 ligands. Correspondingly, blocking the mTOR pathway by peripheral administration of rapamycin in rats prevented the cognitive deficit induced by the injection of 5HT6 receptor agonist in various tests and in developmental models of schizophrenia. Our preliminary data also showed that expression of the 5-HT6 receptor in neuroblastoma cell lines (SH-SY5Y, NG108-15) induced drastic morphological changes usually associated with neuronal differentiation, which are mediated by some of the proteins identified as receptor partners by proteomics. This study highlights the power of proteomics screen to identify novel signalling pathways underlying the physiological functions engaged by transmembrane receptors and physiopathological processes. Link: http://www.fpp.cnrs.fr/
97/381
[P11] Development of PSAQ-SRM assay for IGF-1 quantification in serum

Guillaume Picard1, Michel Jaquinod1, Jrme Garin1, Christophe Bruley1, Virginie Brun1
1
Laboratoire d'Etude de la Dynamique des Protomes, Unit de Biologie Grande Echelle, CEA/INSERM/UJF 1038, 17, rue des Martyrs, 38054 cedex 9 Grenoble, France Contact: Michel Jaquinod (michel,jaquinod@cea.fr)
Keywords: Clinical proteomics, Proteins, peptides and small molecules quantification Insulin-like growth factor 1 (IGF-1) is a 7.7 kDa polypeptide hormone which mediates most effects of growth hormone. In the clinical laboratory, IGF-1 is commonly quantified in blood samples using antibody-based assays with radioactive detection (RIA, IRMA). In addition to the danger posed by radioactive isotopes, these assays lack reproducibility due to the low purity of the calibration standard and due to IGF-1 binding to its carrier proteins. Thus, technological developments for safe and reliable IGF-1 dosage are still required. In this study, we have developed a proteomic assay, based on the PSAQ method (Protein Standard Absolute Quantification) [Brun et al, Mol Cell Proteomics, 2007] and Selected Reaction Monitoring (SRM) analysis, to quantify IGF-1 in serum samples. First, we optimized the expression, isotope-labelling and purification of IGF-1 to serve as quantification standard (PSAQ standard). Then, using serum samples spiked with IGF-1 PSAQ standard, we assessed several prefractionation methods, including abundant protein depletion, for their capacity to enrich IGF-1. After prefractionation and digestion with trypsin, samples were analyzed using microLCSRM. At last, IGF-1 titration was performed in healthy and artificially spiked serum samples to assess the performances of our PSAQ-SRM assay. In conclusion and according to our results, PSAQ-SRM assays hold great promise as surrogates to radioimmunoassays for the dosage of polypeptides hormones and related biosimilars.
98/381
[P12] Optimized systems for stable isotope-labelled protein expression

Dorothe Lebert2, Guillaume Picard1, Cline Huillet1, Nicolas Mouz2, Jrme Garin1, Christophe Bruley1, Virginie Brun1
1
Laboratoire d'Etude de la Dynamique des Protomes, Unit de Biologie Grande Echelle, CEA/INSERM/UJF 1038, 17, rue des Martyrs, 38054 cedex 9 Grenoble, France
2
Promise Advanced Proteomics, 7, parvis Louis Nel, 38040 cedex 9 Grenoble, France
Contact: Virginie Brun (virginie.brun@cea.fr)
Keywords: Proteins, peptides and small molecules quantification Absolute quantification of proteins in complex matrices relies on the use of isotope-dilution standards. Stable isotope-labelled peptides are widely used by the proteomics community but recently, the use of full-length stable isotope-labelled proteins (PSAQ standards) has demonstrated several specific advantages [Brun et al, J Proteomics, 2009]. In particular, as PSAQ standards and protein analytes behave similarly during prefractionation and digestion, a highlyaccurate quantification can be obtained. In this study, we have optimized different expression systems for the high-yield synthesis and isotope-labelling of proteins. These expression systems include: 1. An E. coli lysate for cell-free expression, 2. An E. coli strain, auxotrophic for arginine and lysine biosynthesis. A target protein was expressed and labelled on arginine and lysine residues using these 2 optimized systems. After protein purification and trypsin digestion, isotope incorporation was checked using LC-SRM. Both systems allowed high-levels of protein expression and isotope incorporation (>99%). The relative costs for the production of this labelled protein were also calculated and compared. Presently, we are optimizing a HEK mammalian cell expression system which will allow the synthesis and isotope-labelling of glycosylated proteins. In conclusion, these optimized systems will allow us to adress the expression and isotopelabelling of a large panel of proteins with high success rates.
99/381
[P13] Analyse protomique Caenorhabditis elegans.

1
de
la
rponse
anti-fongique
du
nmatode
Matthieu Pophillat1, Patrick Fourquet1, Jonathan Ewbank1, Carole Couillault1 Centre d'Immunologie de Marseille Luminy U631, 163 avenue de Luminy case 906, 13288 Cedex 9 Marseille, France Contact: Matthieu Pophillat (pophillat@ciml.univ-mrs.fr)
Keywords: Systems biology Lorsque le nmatode Caenorhabditis elegans est infect par le champignon nmatophage Drechmeria coniospora, il est capable dactiver un systme de dfense inn qui permet la synthse de peptides antimicrobiens (Coullault et al, 2004). La dcouverte de ces peptides et les voies de signalisation qui rgulent leur expression ont principalement t mises en vidence par des approches de transcriptomique, de mutagnse et dARN interfrence (ex. Zugasti et Ewbank, 2009 ; Dierking et al, 2011). Pour trouver de nouveaux acteurs impliqus dans les dfenses innes de C. elegans nous avons ralis par 2D-DiGE une tude comparative des protomes de C. elegans infects ou non par D. coniospora. Travailler sur un organisme entier est complexe pour lextraction qualitative et quantitative des protines. Pour augmenter la sensibilit de nos rsultats nous avons opt pour un fractionnement des extraits protiques de nmatode pour obtenir 3 fractions solubles et 1 non-soluble. Cela nous a permis didentifier par MS au total 691 protines dans les diffrentes fractions. Seulement 58 protines ont t trouves comme diffrentiellement reprsentes suite linfection. Leur caractrisation a permis une meilleure comprhension des dfenses innes face linfection fongique.
100/381
[P14] Proteomic profiling of oil-bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved in lipid metabolism
Mai Nguyen1, Mathieu Baudet2, Stphan Cuin1, Jean-Marc Adriano1, Damien Barthe2, Emmanuelle Billon1, Fred Beisson1, Myriam Ferro2, Yonghua Li-Beisson1
1
Laboratoire de bionergtique et biotechnologie des bactries et microalgues, SBVME,IBEB, DSV, CEA Cadarache, CEA Cadarache, 13108 Saint Paul lez Durance
2
Laboratoire dEtude de la Dynamique des protomes, BGE, iRTSV, CEA Grenoble, 17 rue des Martyrs, 38000 Grenoble Contact: Mathieu Baudet (mathieu.baudet@cea.fr)
Keywords: High mass and high resolution mass Spectrometry, Systems biology, Bioinformatics et biostatistics dedicated to proteomics Oil-bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward understanding oil accumulation in algae, we used proteomics to analyze purified oil-bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (2 peptides) identified by LC-MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl-lipids and sterols). Compared to a recently reported Chlamydomonas oil-body proteome, 19 newly identified proteins of lipid metabolism were present, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol 3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated to the oil body fraction. This dataset thus provides evidence that Chlamydomonas oil-bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil-bodies in microalgal cell functions.
101/381
[P15] Amino acid analysis based on isotope-dilution and high-resolution mass spectrometry for sensitive protein quantification
Mathilde Louwagie1, Sylvie Kieffer-Jaquinod1, Yohann Cout1, Michel Jaquinod1, Alain Dupuis1, Jrme Garin1, Christophe Bruley1, Virginie Brun1
1
Laboratoire d'Etude de la Dynamique des Protomes, Unit de Biologie Grande Echelle, CEA/INSERM/UJF 1038, 17, rue des Martyrs, 38054 cedex 9 Grenoble, France Contact: Mathilde Louwagie (mlouwagie@cea.fr)
Keywords: Systems biology Over the last decade, stable-isotope labelled standards (peptides or proteins) have been increasingly used for protein absolute quantification using mass spectrometry. High-quality standards are critical for reliable quantification experiments. Critical features include purity degree, isotope incorporation yield and accurate quantification. Presently, the reference method for standard quantification is amino acid analysis (AAA). AAA analyses are classically based on: (1) an acid hydrolysis of peptides or proteins into amino acids, (2) an amino acid separation using liquid or gas chromatography, (3) a fluorescence detection after amino acid derivatization which can be performed before or after chromatography (1). Traditional AAA analyses are time-consuming (>24h) and require significant amounts of material (around 100g for a single protein quantification). Driven by important needs for the calibration of stableisotope labelled protein standards (PSAQ standards), we have developed a new AAA assay avoiding both derivatization and chromatographic separation of amino acids. This method is based on a microwave-assisted hydrolysis followed by high-resolution mass spectrometry analysis of amino acids, including isotopically-labelled amino acids. Quantification is achieved by sample standardization with isotope-labelled amino acids or BSA standard spiked just before hydrolysis. Our method was evaluated for the calibration of isotopically-labelled proteins (PSAQ standards) and peptides (AQUA standards). Very sensitive (only 1g of protein is needed per analysis), rapid and accurate, this method should be useful in several domains including biotechnology and quantitative proteomics.
102/381
[P16] Quantification of wheat allergens in complex matrices by mass spectrometry

Hlne Rogniaux1, Marion Hamon1, Audrey Geairon1, David Ropartz1, Sandra Denery-Papini1, Colette Larr1
1
INRA Unit BIA, Rue de la Graudire, 44316 NANTES
Contact: Hlne Rogniaux (helene.rogniaux@nantes.inra.fr)
Keywords: Proteins, peptides and small molecules quantification Wheat is an important part of the daily diet of millions of people. Wheat products are consumed in various forms including many kinds and types of breads, cakes, pastas, pizzas and confectionary items. However, this staple food is also responsible for food allergies. Populationbased studies indicate prevalence as high as 0.5% in children, on the basis of positive wheat challenge tests, and high prevalence of sensitization to wheat in adults (assessed by IgE). Hypersensitivity reactions to wheat flour occur both by inhalation (baker's asthma) and ingestion (food allergy and celiac disease), but may also develop by contact in some cases. Clinical symptoms can be severe, leading to anaphylaxis. Many wheat proteins have been reported as causative food allergens, including some prolamintype gluten proteins and salt soluble proteins of the albumin/globulin (A/G) type. The objective of this work was to investigate the ability of mass spectrometry-based methods to quantify previously identified allergens, in complex fractions of wheat proteins. Advantage of these methods, compared to immune detection of allergens by ELISA assays, is that they enable the concomitant monitoring of several allergens within a same protein fraction. In addition, these methods can be used to quantitatively monitor proteins that undergo modifications, for instance in the course of an industrial process: this is clearly more tedious with antibodies, since modification can alter recognition of the epitope. The MS-based quantitative monitoring of wheat allergens was implemented onto a LTQOrbitrap Velos mass spectrometer. A MS/MS method, with quantification from the signal of both the parent and the fragments generated in the linear ion trap, was compared to quantification from the parent signal only, recorded in SIM mode (single ion monitoring) with a high resolution in the Orbitrap. Analytical performances (limit of detection, repeatability, standard deviation, sensitivity, etc.) and experimental difficulties encountered in this study are discussed. Quantification of serpin and alpha-amylase inhibitors, both proteins identified previously as major wheat allergens, could be successfully achieved at low level of abundance in complex grain proteins extracts.
103/381
[P17] Integration between environmental stress and growth in plants

Delphine Cast1, Milena Mozzo1, Christophe Robaglia1, Marie-Hlne Montan1, Benot Menand1
1
Laboratoire de Genetique et Biophysique des Plantes, IBEB, UMR6191 CNRS-CEA-Universit Aix Marseille, 163 avenue deLuminy, 13006 Marseille Contact: Benot Menand (benoit.menand@univmed.fr)
Keywords: Signaling, interactomics and post-translational modifications Our laboratory uses selected model plants like the vascular plant Arabidopsis thaliana and the bryophyte Physcomitrella patens to characterize molecular mechanisms that integrate environmental changes and plant growth, and to understand how they have evolved through land plant evolution. We are focusing our work on the eukaryotic TOR (Target Of Rapamycin) / S6 kinase signaling pathway. Those kinases are central regulators of eukaryotic growth in response to environmental stresses that integrates different environmental signals to control growth and development. TOR and S6K genes are conserved in all eukaryotes but little is known about their function in plants. We will present the recent advancements on our analysis of Physcomitrella patens S6K mutants made in our laboratory. Our results suggest a function of P. patens S6 kinases in the control of plant cell growth and proliferation in response to environmental stresses. We are know starting a proteomic approach to characterize the target of these kinases and to determine how those cell growth phenotypes are associated with essential cellular activities such are protein synthesis. We are particularly focusing our work on the proteomic analysis of ribosomes and translation initiation complexes. Link: http://www.lgbp.univ-mrs.fr/rubrique.php3?id_rubrique=14
104/381
[P18] Etude de ltat doligomrisation de chimiokines induit par des glycosaminoglycanes par Electrophorse Capillaire dAffinit couple la Spectromtrie de Masse Electrospray (ACE-ESI-MS)
Mehdi Prull-Janssen1, Florence Gonnet1, Cdric Przybylski1, David Bonnaff3, Yael Hersant3, Rgis Daniel1
1
Universit Evry-Val-dEssonne, Laboratoire Analyse et Modlisation pour la Biologie et lEnvironnement, bd F. Mitterrand, 91025 Evry, France
2
CNRS, UMR 8587, Laboratoire Analyse et Modlisation pour la Biologie et lEnvironnement, bd F. Mitterrand, 91025 Evry, France
3
Universit Paris Sud 11, ICMMO, Laboratoire de Chimie Organique Multifonctionnelle, rue G. Clmenceau, 91405 Orsay, France
4
CNRS UMR 8182, ICMMO, Laboratoire de Chimie Organique Multifonctionnelle, rue G. Clmenceau, 91405 Orsay, France Contact: Florence Gonnet (florence.gonnet@univ-evry.fr) Keywords: Signaling, interactomics and post-translational modifications, Separative analysis methods, Systems biology Ltude des interactions entre polysaccharides et protines est dun intrt majeur en glycobiologie, puisque les complexes rsultants sont impliqus dans des processus biologiques fondamentaux. L'identification d'un ligand glycosidique spcifique dune protine reste une tche difficile en raison de l'immense diversit structurale des oligosaccharides. Afin de surmonter ces difficults, nous avons mis en place le couplage en ligne de l'lectrophorse capillaire daffinit (ACE) avec la spectromtrie de masse (MS) electrospray (ACE-ESI/MS). Ce couplage est un outil analytique puissant pour la caractrisation de complexes non-covalents entre les protines et les glycosaminoglycanes (GAG) (1). La composante ACE permet (a) de caractriser linteraction via les constantes de dissociations (Kd), et (b) disoler le ligand glycosidique. La composante ESI/MS permet de caractriser les stchiomtries des complexes non-covalents. Nous avons ainsi pu tudier l'effet d'oligosaccharides sulfats de synthse sur ltat doligomrisation de la chimiokine MCP-1 (Monocyte Chemoattractant Protein 1), ainsi que leur spcificit de liaison. Le caractre basique de MCP-1 (pI 9.74) a ncessit le greffage dhydroxypropylcellulose (HPC) sur la paroi interne du capillaire afin de limiter ladsorption de la protine. Ce greffage est compatible avec la MS. Les interactions MCP-1/GAGs ont t tudies en utilisant six tetrasaccharides et un hexasaccharide synthtiques dhparine, variant par le nombre et la position des groupements sulfate. La migration lectrophortique de MCP-1 a t mesure en prsence de diffrentes concentrations doligosaccharides dans llectrolyte, afin de dterminer les Kd des complexes. Tous les paramtres de couplage ont t optimiss pour prserver lintgrit structurale des complexes non-covalents. Nos rsultats montrent quen solution la chimiokine MCP-1 est monomrique. En revanche, en prsence doligosaccharides sulfats, nous observons loligomrisation de MCP-1, comme prcdemment observ pour SDF-1 (3). En utilisant des GAGs synthtiques, lACE-ESI/MS a permis de mettre en vidence limportance la fois de la sulfatation (nombre et position des groupements sulfate) mais aussi de la taille de loligosaccharide sur le degr doligomrisation de MCP-1. 1- S. Fermas et al. Anal Chem, 2007, 79, 4987-4993 2- S. Fermas et al. Anal Biochem, 2008, 372, 258-260 3- S. Fermas et al. Glycobiology, 2008, 18, 1054-1064 Link: http://www.lambe.univ-evry.fr/
105/381
[P19] Relative quantification of wax esters in complex lipid mixtures

Laetitia Fouillen1, Franziska Dittrich-Domergue1, Ren Lessire1, Frderic Domergue1
1
Laboratoire de Biogense membranaire, Universit Sgalen, CNRS, UMR 5200, 146, rue Lo Saignat, 33076 Bordeaux, France Contact: Laetitia Fouillen (laetitia.fouillen@u-bordeaux2.fr)
Keywords: Proteins, peptides and small molecules quantification Wax esters are very long hydrophobic molecules made of a fatty acid and a fatty alcohol. In plants they are a part of the cuticle, a protective layer covering all aerial parts. Wax esters are much more resistant to high temperatures and pressures than normal plant oils, so that they represent an interesting class of lipids for industry, in particular as lubricants. The goal of the ICON project is to generate plants that produce wax esters in seeds instead of classical triglycerides. The biosynthesis of wax esters is a two steps pathway: first a Fatty Acid Reductase reduces a fatty acid (RCOOH) into an alcohol (RCH2OH); second, a Wax Ester Synthase esterifies another fatty acid (RCOOH) onto the alcohol to form the wax ester (RCH2OCOR). We induced the production of wax ester in yeast by co-expressing the two enzymes. This model produces C30 to C36 wax esters. The goal of the present study was to develop a sensitive and powerful method to quantify complex mixtures of wax esters. Wax esters are usually identified by GC-EI-MS. Nevertheless, multiple isobaric components are not separated chromatographically making quantification problematic. To avoid theses drawbacks and to quantify all components, we developed a LCMS/MS method using a liquid chromatograph (Dionex) with reverse-phase column and QTrap mass spectrometer (Applied Biosystem). Data were acquired using MRM mode. A C42 synthetic wax ester was used as internal standard to allow quantification. Preliminary results show that our LC-MS/MS method allows the identification and relative quantification of wax esters components with a better sensibility compared to GC-MS analysis. This method should allow us to analyze the different wax esters produced in plants
106/381
[P20] Tandem mass spectrometric analysis of a mixture of isobars using the survival yield technique.
Antony Memboeuf1, Laure Jullien1, Remi Lartia1, Bernard Brasme1, Yves Gimbert1
1
Departement de Chimie Moleculaire, 301 rue de la Chimie, 38041 Grenoble Cedex 9, France
Contact: Antony Memboeuf (a.memboeuf@laposte.net)
Keywords: Separative analysis methods, Proteins, peptides and small molecules quantification , Organic and inorganic mass spectrometry There is a growing demand on mass spectrometric techniques for high-throughput analysis, to perform more reliably and provide more information on increasingly complex samples: the analysis of compounds in biological samples, in the field of petroleomics, to study isomers or simply for separating unresolved peaks due to technical limitations. With this respect, the identification and quantification of isobars remain challenging tasks for mass spectroscopists. The identification and, possibly, elimination of an isobar becomes essential in order to perform the structural analysis, ultimately to quantify, a compound of interest. This may require a tedious preparative work, coupling with chromatographic separation techniques or more recently ion mobility spectrometry, possibly combined with complex procedures. Collision Induced Dissociation tandem mass spectrometry experiments were performed to unequivocally separate compounds from an isobaric mixture of two products. The Survival Yield curve was obtained and, is shown to consist in a linear combination of the curves corresponding to the two components separately. For such a mixture, a plateau appears on the diagram in lieu of the continuous decrease expected allowing for the structural study of the two components separately. The width of the plateau critically relates to the fragmentation parameters of the two molecular ions, which need to be structurally sufficiently different for the plateau to be observed. However, at constant fragmentation parameters, we have observed the width significantly increases at large m/z. This makes the separation more and more efficient as isobars have larger m/z and the technique complementary to those applicable at low m/z only. We have observed the vertical position of the plateau relates linearly to the relative concentration of the two compounds that may be useful for quantification. Repeatability was estimated at 2% on a quadrupole ion trap. An advantage of using Survival Yield curves only, is that a priori knowledge of the respective fragmentation patterns of the two isobars becomes unnecessary. Consequently, similar performances are obtained if fragments are isobaric, which is also demonstrated in our study. Using this methodology, structural study and quantification of a compound of interest can be performed without the need for cleaning the sample from the isobaric contaminant.
107/381
[P21] Stratgies de prparation dchantillon en ligne en vue de lanalyse rapide de composs pharmaceutiques et cliniques dans des matrices biologiques par chromatographie liquide coupl la spectromtrie de masse (LC-MS/MS)
Emmanuel Bourgogne1, Olivier Laprvote2, Jean Louis Beaudeux2, Emmanuel Varesio1, Grard Hopfgartner1
1
Groupe de Spectromtrie de masse du vivant, Ecole de Pharmacie, Universit Genve, Universit Lausanne, 20 Bd dYvoy, 1204 Genve, Suisse
2
Laboratoire de Toxicologie Biologique, Hpital Lariboisire, Assistance Publique-Hpitaux de Paris, 2 rue Ambroise Par, 75010 Paris, France
3
Laboratoire Chimie-Toxicologie Analytique et Cellulaire, EA 4463, Facult des Sciences Pharmaceutiques et Biologiques, Universit Paris Descartes, 4 avenue de LObservatoire, 75006 Paris Contact: Emmanuel Bourgogne (emmanuel.bourgogne@parisdescartes.fr)
Keywords: Proteins, peptides and small molecules quantification , Separative analysis methods La ncessit d'laborer des stratgies gnriques pour cribler rapidement et/ou de quantifier de nombreux composs dans lindustrie pharmaceutique ou les laboratoires de toxicologie biologique demeure un dfi. Aujourdhui, les stratgies modernes de bioanalyse vont de la simple dilution un prtraitement des chantillons complexes, comme l'extraction en ligne en phase solide (SPE) ou l'extraction liquide-liquide (LLE). Pour acclrer le dveloppement de mthodes et leur validation, les approches gnriques injection directe de fluides biologiques sont hautement souhaitables. Le Prospekt 2, un dispositif de SPE en ligne, a t valu comme un systme polyvalent de commutation de colonne avec une dilution en ligne et des injections rptes sur la mme cartouche de pigeage. Les objectifs de ce travail sont de prsenter, travers diffrents exemples, l'avantage d'utiliser le mme matriel, le systme Prospekt, comme un dispositif de prparation de l'chantillon soit en ligne, soit hors ligne. Sa flexibilit est mise en vidence lorsque la rtention sur le matriel de pigeage ou le transfert sur la colonne analytique est problmatique, en utilisant respectivement une dilution en ligne pr-/ ou postcolonne de pigeage. Sa facilit d'utilisation pour changer les cartouches de diffrentes chimies complte les outils Prospekt pour faire face tous les analytes pour le dveloppement de mthode gnrique et la quantification de petites molcules dans les liquides biologiques. Enfin, dans le but d'amliorer le dbit danalyse lors du suivi thrapeutique mdicamenteux (STM), la SPE en ligne couple une chromatographie liquide et un dtecteur de masse de type trappe linaire (LC-MS/MS) a t value. Dans ce cadre du STM, une approche gnrique est ainsi propose pour estimer la concentration de nombreux mdicaments dans des chantillons biologiques. Cet appareil peut donc tre utilis comme un one size fits all" instrument capable d'effectuer diverses stratgies en ligne ou hors ligne. Une stratgie adapte sera choisie en fonction des proprits physico-chimiques du compos d'intrt, de la sensibilit de la mthode ncessaire ainsi que du dbit danalyse souhait.
108/381
[P22] Stringent evaluation of several label free quantitative workflows using a complex protein standard and statistical analysis
Emmanuelle Mouton-Barbosa1, David Bouyssi1, Stphanie Bolot1, Florence Roux-Dalvai1, Bernard Monsarrat1, Anne Gonzalez de Peredo1, Odile Burlet-Schiltz1
1
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, Universit de Toulouse, 205, route de Narbonne, 31077 BP64182 Toulouse, France Contact: Emmanuelle Mouton-Barbosa (emmanuelle.mouton@ipbs.fr)
Keywords: Proteins, peptides and small molecules quantification , Bioinformatics et biostatistics dedicated to proteomics To study differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Label free methods offer many advantages over isotopic labeling approaches: they are easy to use at the sample preparation step, cheaper, applicable to any type of samples, and they allow the comparison of a high number of conditions, opening the way to clinical studies. However, they also require complex bioinformatic MS data processing, such as peak picking, realignment, normalization, and peptide peaks matching across different nanoLC-MS runs. Many bioinformatic tools that have been developed in recent years for label-free quantification use peptide pattern-based methods, in which the software performs feature detection in LC-MS maps through analysis of the characteristic isotopic pattern of a peptide ion in the m/z dimension, and of its chromatographic elution peak in the retention time (RT) dimension, and links afterwards this quantitative measurement to a peptide identification. On the other hand, another approach is based on the reverse process, i.e. making use initially of peptide identification results to go back in the MS scans in order to extract peptide intensity values, using experimentally measured m/z and RT as a starting point to retrieve the associated extracted ion chromatograms (XIC) of this ion. Here we evaluated three label-free workflows for the quantitative analysis of highly complex protein mixtures, using both strategies: our in-house developed software MFPaQ [1-2], which starts from identification results, and two other label-free programs, LCprogenesis and MSInspect, that were used to generate LC-MS peptide maps. Our new in-house software Prosper allowed to integrate the quantitative data extracted with each tool, and to link these quantitative results with validated identified proteins. In order to perform an unambiguous evaluation of these quantitative workflows in the context of a real differential study, we used an equimolar mixture of 48 human proteins (Sigma UPS1), spiked at different concentrations into a lysate of SILAC-labeled U937 cells. Samples were run in triplicate on an LTQ VELOS-Orbitrap mass spectrometer, and statistical analysis was performed to identify differential peptides and proteins for theoretical fold variations of 2, 10 or 100 of the spiked standards. This experimental design allowed us to determine the number of true and false-positive (respectively UPS1 or U937 background proteins found as differential), and we present the performances of each workflow in terms of sensitivity, specificity, and accuracy. 1- Bouyssie, D., et al Mol Cell Proteomics, 2007 2- Mouton-Barbosa, E., et al. Mol Cell Proteomics, 2010
109/381
[P23] ICM-MS and Top down analysis to characterize biomarker

Valrie Labas1, Grgoire Harichaux1, Chrystelle Derache3, Thierry Magallon2, Ana-Paula Teixeira-Gomes1, Maxime Coulon3, Anne-Christine Lalmanach3
1
INRA, Plate-forme dAnalyse Intgrative des Biomarqueurs, Centre de Recherches INRA de Tours, 37380 Nouzilly
2
INRA, UMR INRA 85 Physiologie de la Reproduction et des Comportements UMR CNRS 6175 Universit de Tours - IFCE, Centre de Recherches INRA de Tours, 37380 Nouzilly
3
INRA, UR1282, Infectiologie Animale et Sant Publique, Centre de Recherches INRA de Tours, 37380 Nouzilly
Contact: Valrie Labas (valerie.labas@tours.inra.fr)
Keywords: Signaling, interactomics and post-translational modifications, Separative analysis methods, Systems biology Intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) is a well established differential and quantitative approach to display biomarkers on prokaryotic or eukaryotic cells. ICM-MS is a fast, reproducible and sensitive method for peptidomic and proteomic studies in 2-30 kDa mass range. However, by this approach the biomarker of interest remain to be identified. Here we demonstrate the application of ICM-MS profiling coupled to Top-down analysis to characterize three avian -defensins : AvBD1, AvBD2 and a newly isolated -defensin AvBD7. -defensins are important components of chicken innate immunity in mucosal tissue, a major entry site for pathogens that may cause food poisoning. Chicken inbred lines diverge phenotypically with respect to levels of pathogens intestinal carriage and to level of gene expression of two -defensin (AvBD1, AvBD2). In order to detect the known mature -defensins and to discover other molecular species with a potential antimicrobial activity, ICM-MS was performed on intact heterophil granulocytes, which are known to infiltrate intestinal tissue early after pathogen colonization. The in silico predicted AvBD1 and AvBD2 sequences, taking into account the three characteristic -defensins disulfide bonds, were compared to experimental peaks. Despite the lack of precision mass in linear MALDI-TOF spectra, two peaks may correspond to AvBD1 and AvBD2. To characterize finely molecular species observed on ICM-MS profile, we purified them by chromatography from bone marrow extract, in which defensin genes have been shown to be highly expressed and that contains the heterophils precursors. All size exclusion chromatography fractions were analysed by classic bottom-up proteomic in order to identify molecular species. AvBD1 and AVBD2 and a newly isolated AvBD7 were identified. To obtain structural information, a Top-down proteomic approach was applied on RP-HPLC purified intact molecular species. By a top-down MS strategy using nano-ESIQ-TOF MS, the AvBDs were measured with a mass accuracy of 100 ppm. By comparison with the theoretical monoisotopic mass (with 3 disulfide bonds), bone marrow AvBD2 exhibited the expected mass but AVBD1 and AvBD7 presented respectively a delta mass = -1 Da and -17 Da. Through the topdown MS/MS strategy, we confirmed respectively the C and N terminal sequences for AvBD1 and AvBD7 and characterized post-translational modifications which correspond to an amidated C-terminal and to a Gln N-terminal cyclized as a pyroglutamic acid residue. In this study, a hybrid strategy combining ICM-MS, bottom-up and top-down proteomic approaches enabled, for the first time, characterization of AvBD1, AvBD2, and AvBD7 as well as isoforms of these defensins. The use of complementary ICM-MS Top-down technologies allowed us to follow these structures in their biological (or cellular) context.
110/381
[P24] Human cell-line phosphoproteome mapping using a dual-funnel ETD ion trap
Yann Hbert1, Chia-feng Tsa2, Pierre-Olivier Schmit1, Andrea Kiehne3
1 2 3
BRUKER, 34 rue de l'Industrie, 67166 WISSEMBOURG Institute of Biomedical Sciences, Taipei 115, TAIWAN, China Bruker Daltonik GmbH, Fahrenheit Strasse, Bremen, Germany
Contact: Yann Hbert (yann.hebert@bruker.fr)
Keywords: Signaling, interactomics and post-translational modifications, Instrumentation One of the essential regulatory mechanisms in eukaryotic cell is driven by the action of kinases. They affect the information flow through the signaling processes and consequently exert an influence on cells, tissues and organisms phenotype and functions. Altered regulations of these processes are observed in a variety of diseases, among which many cancers. Therefore, detecting, characterizing and ultimately quantifying the phosphoproteins altered in these pathologies is an important step towards a better understanding of the implicated processes. The low concentration level and ionization efficiencies of these compounds makes it difficult to analyze them directly by mass spectrometry while in mixture with other cellular proteins at their natural ratio We are presenting here the results obtained after human cell line IMAC phosphoprotein enrichment followed by an ESI CID/ETD Auto MSn experiment. The purpose was both to assay enrichment protocol efficiency and to check the ability of the LC-MS/MS ETD System to map reproducibly the phosphorylation sites. Three technical replicates have been analyzed, yielding the identification of 95 proteins, 88 of them being identified in each of the three runs. Among these, 93 are phosphoproteins and 87 of them, representing 220 phosphorylated peptides have been found in the three replicates. The high quality of the obtained CID and ETD spectra combined with the use of dedicated bio informatics has enabled to map efficiently the phosphorylation sites for most of these peptides. We are now ready to use this technique on a higher to proceed to a more systematic mapping of the phosphoproteins present in our sample.
111/381
[P25] UPLC-ESI-MS study of the oxidation markers released from tannin depolymerisation: toward a better control of the tannin evolution over wine production.
Laetitia Mouls1, Stphanie Carrillo 1, Ana-Beln Bautista-Ortn1, Hlne Fulcrand1
1
Laboratoire Sciences pour l'nologie UMR1083, INRA, Montpellier SupAgro, Universit Montpellier 1, 2, place Pierre Viala, 34060 Montpellier, France Contact: Laetitia Mouls (laetitia.mouls@supagro.inra.fr)
Keywords: Organic and inorganic mass spectrometry Among the numerous compounds occurring in wine, condensed tannins take an important part in the sensory quality of the wine; they are thus responsible for astringency, bitterness and contribute to colour as well. Tannins, also called proanthocyanidins, are very reactive biopolymers, specifically toward oxidation. The reaction takes place with dissolved oxygen and a catalyst (mainly oxidases in the course of wine making and metal ions over storage). Two reaction pathways were established1. The first one directly involves the phenolic compounds in the oxidation process. The other one involves other wine constituents such as ethanol and hydroxy-acids, yielding aldehydes that then react with tannins and anthocyanins. The resulting pigments have a positive impact in the wine colour while other oxidation products may lead to colloidal instability. Given the intrinsic complexity of tannin structures, their direct oxidation products have not been yet characterized. Their identification is still a challenge due to the heterogeneity of the polymerisation degrees within wine tannins which is enhanced by the large structural diversity arising from oxidation. An UPLC-ESI-MS method was thus developed in order to identify oxidized tannins in wine samples. Given the difficulties to separate and detect tannins with high degrees of polymerisation (DP) by mass spectrometry2, samples were depolymerised by chemical depolymerisation prior to UPLC-ESI-MS analysis. Since the linkages created by oxidation are not cleavable in the usual depolymerisation conditions contrarily to interflavanic linkages, specific oxidation residues should be released from tannins structures after their oxidation. Furthermore, oxidation involving two tannins subunits of two different polymeric chains, which are intermolecular reactions, are supposed to give dimeric structures containing biphenyl-type linkage whereas reactions occurring between two subunits of the same polymeric chain, which are intramolecular reactions, are expected to give dimeric structures containing A-type linkage. First experiments carried out on procyanidin B2 after oxidation allowed both intramolecular and intermolecular oxidation markers to be clearly identified. This primary study was completed by other analyses carried out on several apple tannin fractions (tannins solely composed of a variable number of epicatechin subunits) after oxidation and other types of markers were identified. Analyses of tannin fractions extracted from wines are currently under way so as to identify and quantify wine oxidation markers and to better understand the relationships between phenolic composition and wine quality. [1] H. Fulcrand, M. Duenas-Paton, E. Salas, V. Cheynier, Am. J. Enol. Vitic. 57 (2006) 289-297. [2] L. Mouls, J-P. Mazauric, N. Sommerer, H. Fulcrand and G. Mazerolles, Anal. Bioanal. Chem. 400 (2011) 613-623.
112/381
[P26] From single cell MALDI-TOF profiling to protein identification in mammalian egg: integrative approach to study the oocyte quality
Svetlana Uzbekova1, Lucie Spina1, Ana-Paula Teixeira-Gomes1, Valrie Labas1
1
INRA, Plate-forme dAnalyse Intgrative des Biomarqueurs, Centre de Recherches INRA de Tours, 37380 Nouzilly, France
2
INRA, UMR INRA 85, Physiologie de la Reproduction et des Comportements UMR CNRS 6175 Universit de Tours -IFCE, Centre de Recherches INRA de Tours, 37380 Nouzilly, France
3
INRA, UR1282, Infectiologie Animale et Sant Publique, Centre de Recherches INRA de Tours, 37380 Nouzilly, France Contact: Svetlana Uzbekova (Svetlana.Uzbekova@tours.inra.fr)
Keywords: Systems biology, Imaging mass spectrometry Capacity of female germ cell, an oocyte, to be fertilized and to develop to viable embryo is oocyte quality, which is acquired throughout meiotic maturation during pre-conceptual period in mammals. A number of proteins are produced in oocyte during this period to assure early development of future embryo. Mammalian oocyte is 100-150 microns in diameter and contain about 80 ng of protein, thus classic differential proteomics between immature and mature oocytes required thousands of eggs. We adapted Intact Cells MALDI-TOF Mass Spectrometry (ICM-MS) approach on single bovine oocyte by using MALDI-TOF mass spectrometer operating in positive linear mode. Therefore we obtained and compared the spectra profiles as fingerprints of different oocytes. Objective was to look for differential proteins between the oocytes that were able (mature) or not (immature) to be fertilized. Reproducible mass fingerprint gathering a number of peaks ranged 3 000 25 000 m/z were obtained for groups of immature and mature oocytes (n=30). Intensity peak variations were observed for 40 from 114 of detected peaks between the oocytes of two groups (ANOVA, p<0.01, Progenesis-MALDI, Non-Linear Dynamics). Among them intensity of 14 peaks varied more than two fold. In order to identify the differential proteins between immature and mature oocytes, we performed nano-LC-MSMS of proteins resolved by 1D-SDS-PAGE. 337 different bovine proteins were identified, among them 149 were common for immature and mature oocytes. Unfortunately, the identification of polypeptides from separated extract by SDS-PAGE does not allow the direct attribution of a candidate to a peak. By too many changes (adding post-translational chemic groups on fragments associated or not with proteolysis) no correspondence can be made between the theoretical mass and the experimental mass. However, comparison of m/z values of differential peaks with predicted molecular weight of identified proteins allowed constituting the list of potential candidates, which may to be validated by immunodetection methods. For better identification of protein origin of differential peaks, a Top down proteomic strategy consisting of nano-HLPC purification of small size proteins with their direct fragmentation using high resolution mass spectrometry will be committed. In conclusion, intact cell MALDI-MS was adapted to mammalian single oocyte and allowed discrimination between the oocytes of different maturation stages by a number of small size proteins which could be identified and served as potential biomarkers.
113/381
[P27] Biomarkers of chicken sperm quality displayed by Intact Cells MALDI-TOF Mass Spectrometry
Valrie Labas1, Grgoire Harichaux1, Isabelle Grasseau4, Jean-Didier Terlot4, Ana-Paula Teixeira-Gomes1, Xavier Druart2, Nadine Grard2, Elisabeth Blesbois4
1
INRA, Plate-forme dAnalyse Intgrative des Biomarqueurs, Centre de recherches INRA de Tours, 37380 Nouzilly, France
2
INRA, UMR INRA 85 Physiologie de la Reproduction et des Comportements - UMR CNRS 6175 Universit de Tours -IFCE, Centre de recherches INRA de Tours, 37380 Nouzilly, France
3
INRA, UR1282, Infectiologie Animale et Sant Publique, Centre de recherches INRA de Tours, 37380 Nouzilly, France
4
INRA, UR 83 Recherches Avicoles, Centre de recherches INRA de Tours, 37380 Nouzilly, France
Contact: Valrie Labas (valerie.labas@tours.inra.fr)
Keywords: Systems biology Fertility is a complex and highly variable physiological parameter that depends on individual gamete composition and quality. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a rapid and sensitive analytical approach. It measures molecular weights of peptides and proteins < 20 kDa that are present in complex biological samples, such as cells. This approach does not need extensive sample preparation, except for the cell isolation and matrix application. In this study, differential and quantitative analysis using Intact Cells MALDI-TOF Mass Spectrometry (ICM-MS) profiling, was performed on intact chicken ejaculated spermatozoa. Coupled to biological tests, this method was used for a rapid profiling to display potential biomarkers implicated in fertility. Sperm ICM-MS, Sperm viability (dye SyBr14/IP), motility (Computer Assisted Semen Analyses), induction of acrosome reaction (dye PNA/FITC) were examined on the same biological samples. Individual male fertility was also evaluated after a single artificial insemination of 10-12 hens/male. Three ejaculated sperm samples were collected from 12 chickens for the MS analysis. Spermatozoa were washed and suspended in Tris-Sucrose. The whole cells were spotted onto the MALDI sample probe with an aliquot of a sinapinic acid. All mass spectra were recorded using a MALDI-TOF mass spectrometer (M@LDI LR, Waters), operating in positive linear mode. Eighty four spectral profiles were collected in the mass range 2000-20000 m/z with one hundred spectra per well. Data processing was performed using MassLynx software. Alignment, peak intensity detection and statistical analysis (ANOVA and PCA) were done using ProgenesisMALDI software (Nonlinear Dynamics). The analysis by male gave abundant mass spectral information. Among a total of 144 m/z observed, differentially expressed peptides or proteins were established according to a probability value < 0.05. Twenty nine m/z were retained with progressive significant intensities variations. PCA analysis demonstrated variability between animals. Our results showed correlation between spectrum profiles and physiological parameters. We characterized 4 peaks that significantly differentiated males on fertility, 4 on acrosome reaction and 18 on motility. Only one of them was significantly different for all tests. In conclusion, we showed that ICM-MS can be used to profile peptides and proteins from chicken spermatozoa, which could be involved in fertility. Micropurification of molecular species will be considered for Top-down identification with high resolution mass spectrometry.
114/381
[P28] Evaluation de facteurs affectant lanalyse doligosaccharides anioniques par DESI-MS

Cdric Przybylski1, Florence Gonnet1, William Buchmann1, Rgis daniel1
1
Universit Evry-Val-dEssonne, Laboratoire Analyse et Modlisation pour la Biologie et lEnvironnement, bd F. Mitterrand, 91025 Evry, France
2
CNRS, UMR 8587, Laboratoire Analyse et Modlisation pour la Biologie et lEnvironnement, bd F. Mitterrand, 91025 Evry, France Contact: Cdric Przybylski (cedric.przybylski@univ-evry.fr)
Keywords: High mass and high resolution mass Spectrometry, Systems biology, Signaling, interactomics and post-translational modifications Les techniques dionisation actuellement les plus employes en spectromtrie de masse (MS) pour la caractrisation des oligosaccharides et notamment des glycosaminoglycanes (GAGs), sont llectrospray (ESI) et la dsorption ionisation laser assiste par matrice (MALDI)1,2. Rcemment, la dsorption ionisation assiste par lectrospray (DESI)3 a t introduite. Cette mthode requiert un minimum de prparation et prsente une grande tolrance aux sels, permettant lanalyse directe et en conditions atmosphriques ambiantes dchantillons dposs sur une surface. Les GAGs sont des polysaccharides linaires trs anioniques constitus dunits disaccharidiques rptitives. Parmi eux, lhparane sulfate (HS) et lhparine (HP) sont les deux reprsentants les plus emblmatiques des GAGs. Ils comportent de 20 200 units incluant un acide hexuronique et une D-glucosamine actyle et substitue avec des groupements sulfate en position variable. A loppos, lacide hyaluronique (HA) (non sulfat) est structuralement le membre le plus simple de cette famille. De part leur taille, lhtrognit de leurs chanes et la prsence des groupements sulfate trs labiles, les GAGs sont parmi les biopolymres les plus difficiles caractriser4. Impliqus dans de nombreuses fonctions physio-pathologiques, ils agissent notamment par interaction avec des protines cibles5. Les GAGs et leurs assemblages glycoprotiques reprsentent une cible thrapeutique de choix, et ncessite donc une bonne comprhension des proprits la fois structurales et de formation de ces complexes. Nous avons rcemment dmontr que le couplage DESI-MS avec un LTQ-OrbitrapTM permettait de dterminer la composition en oligosaccharides de mlanges dHS et dHP, ainsi que dobserver des complexes non covalents spcifiques avec des protines.6 Dans cette tude, nous valuons diffrents paramtres afin doptimiser la dtection par DESIMS doligosaccharides anioniques. Les limites de dtection ont t dtermines. De plus, des expriences de MSn ont dmontr lefficacit dun tel couplage pour diffrencier des isomres ou encore effectuer un squenage. (1) Zaia, J. Chem. & Biol. 2008, 15, 881-892. (2) Przybylski, C. et al. Glycobiology 2010, 20, 224-234. (3) Takats, Z. et al. R. G. Science 2004, 306, 471-473. (4) Sasisekharan, R. et al. V. Ann. Rev. Biomed. Engin. 2006, 8, 181-231. (5) Gandhi, N.S. et al. Chem. Biol. & Drug Design 2008, 72, 455-482. (6) Przybylski, C. et al. Anal. Chem. 2010, 82, 9225-9233. Link: http://www.lambe.univ-evry.fr
115/381
[P29] The Orbitrap technology: Increased analytical performances for the analysis of peptides and proteins.
Claire Dauly1, Martin Zeller2, Catharina Crone2, Carmen Paschke2, Mathias Mueller2, Eugen Damoc2, Eduard Denisov2, Alexander Makarov2, Bernard Delanghe2, Thomas Moehring2
1 2
Thermo Fisher Scientific, 16 avenue du Qubec, 91963 Courtaboeuf cedex Thermo Fisher Scientific, , Bremen, Germany
Contact: Claire Dauly (claire.dauly@thermofisher.com)
Keywords: Instrumentation Getting a comprehensive view of the protein content of a complex sample is still a challenge in proteomics, getting that done in one analytical run even more. This work will describe new analytical platforms and software with increased performances in terms of scan speed, resolution and sensitivity. A tryptic digest of a whole SILAC labeled HeLa cell lysate was measured on a new Thermo Fisher Scientific benchtop quadrupole Orbitrap instrument coupled to a Thermo Fisher Scientific Easy nano-LC. More than 50.000 HCD spectra were acquired. The raw files were analyzed using Mascot, SEQUEST and X!Tandem. The statistical significance of the identifications was done by applying Percolator to the primary target decoy results of the individual search engines. This resulted in more than 10.000 peptides and more than 2000 proteins identified with less than 1% FDR. Enzymatically degraded E.coli total protein extract was analyzed on a modified Thermo Fisher Scientific LTQ Orbitrap Velos instrument including a compact Orbitrap analyzer and improved pre-amplifier which results in a higher scan speed and improved dynamic range and sensitivity. Proteome Discoverer 1.2 was used for database searching and result validation. The higher scan speed in combination with the better sensitivity results in a higher number of protein and peptide identifications compared to the standard LTQ Orbitrap Velos instrument using the same sample and LC conditions. More precursor ions of low abundant compounds get over the limit of detection and the faster scan rate allows also triggering those precursor ions for MS/MS.
116/381
[P30] Protomique, cellules endothliales et cancer

Bruno Baudin1, Arnaud Bruneel1, Jolle Vinh2, Nelly Bosselut3
1 2 3
EA-4530 UFR Pharmacie UPS, 5 Rue Jean-Baptiste Clment, 92296 Chtenay-Malabry, France USR-3149 CNRS-ESPCI, 10 Rue Vauquelin, 75006 Paris, France
Service de Biochimie A - Ple Biologie-Imagerie Hpital Saint-Antoine, 184 Rue du faubourg Saint-Antoine, 75571 Paris cedex 12, France Contact: Bruno Baudin (bruno.baudin@sat.aphp.fr)
Keywords: Systems biology, Clinical proteomics, High mass and high resolution mass Spectrometry Les cellules endothliales (CE) bordent la face interne des artres, des veines et des capillaires o elles sont linterface du sang et des vaisseaux. Elles exercent de nombreuses fonctions finement rgules, dont des changes avec le sang et les tissus sous-jacents, et les rgulations du tonus vasculaire, de lhmostase, de la fibrinolyse, des rponses inflammatoires et immunitaires. Les CE interviennent dans le cancer sous plusieurs aspects : leur prolifration menant langiogense des tumeurs, la production de facteurs de croissance pour les cellules musculaires lisses de la mdia favorisant la vasculogense tumorale, et leur sensibilit aux chimiothrapies vhicules par le sang amenant des effets indsirables. Nous avons tudi par la protomique, alliant llectrophorse bidimensionnelle et la spectromtrie de masse (MALDITOF et TOF-TOF, LC-ESI-MS/MS, FTICR-MS et visualisation 3D), les CE de la veine ombilicale humaine cultives in vitro (Nature Protocols 2007) dans diverses conditions : (1) au repos confluence, (2) aprs traitement par des mdicaments anticancreux toxiques pour les vaisseaux sanguins, et (3) par des substances mimant langiogense tumorale. Nous avons identifi plus de 600 protines incrimines dans les diverses fonctions des CE (consultables sur notre site huvec.com), dont des protines non connues dans ces cellules ce jour, des protines induites dans lapoptose des CE soumises aux chimiothrapies (Proteomics 2003 et 2005), et des protines incrimines dans langiogense, comme loxygen-regulated protein 150 kDa et des protines chaperons (HSP, GRP, PDI) dont certaines spcifiques aux CE (endoPDI). De ces travaux pourraient merger des nouveaux marqueurs des CE, des marqueurs dapoptose induite par les chimiothrapies anticancreuses, ainsi que des stratgies thrapeutiques pour la protection de lendothlium vasculaire, ou pour inhiber langiogense tumorale. Link: huvec.com
117/381
[P31] Mass spectrometric quantification of trastuzumab, a humanized monoclonal antibody in human plasma
Sylvere Durand1, Keyvan Rezai1, Sophie Weill1, Laetitia Cravello3, Francois Becher2, Francois Lokiec1
1 2 3
Laboratoire Pharmacologie Institut Curie - Hopital Rene Huguenin, 35 rue Dailly, 92210 Saint-Cloud, France CEA Laboratoire d'etude du metabolisme des medicaments, , 91191 Gif-sur-Yvette, France Waters Corporation, , Manchester, United Kingdom
Contact: Sylvere Durand (sylvere.durand@curie.net)
Keywords: Proteins, peptides and small molecules quantification Background: Mass Spectrometer (MS) coupled with Liquid Chromatography (LC) is a very sensitive and selective method for determination of anticancer drug concentrations in different biological matrix. Trastuzumab is a monoclonal antibody that targets the extracellular domain of HER2, a member of the epidermal growth factor receptor (EGFR) family. Trastuzumab is currently approved for the treatment of breast cancer overexpressing HER2. For the quantification of therapeutic monoclonal antibodies in biological specimens, enzyme-linked immunosorbent assay (ELISA) is the most widely used technique. ELISA has some limitations and therefore alternative analytical techniques have to be explored. The aim of this study is to develop a more sensitive analytical method using LC-MS/MS to determine plasma concentrations of trastuzumab with a lower limit of quantification than ELISA. Methods: Method development consisted in several steps: 1- Peptide mapping of trastuzumab is done by enzymatic digestion in collaboration with Waters INC. using a Xevo G2 QToF (Quadrupole Time of Flight) and BiopharmaLynx as software. 2- Peptides potentially candidates to be used as markers are compared to peptides in an online database. The comparison follows the similarity search method (BLAST) to determine the specificity of each peptide obtained from trastuzumab. Other parameters such as ion signal intensity, miscleavage and post-translational modification are included in this analysis for peptide marker targeting. The hypothesis raised by the BLAST method has to be experimentally confirmed. 3- Extraction method using immunocapture with specific antigen/antibody binding is used. Optimal protocol of protein digestion is set up by playing on pH values, solvent ratios, reagent concentrations and trypsin activity duration. 4- Optimization of the coupled Ultra Performance LC and MS QToF G1 Xevo is done with synthesized peptides, similar to the previously selected ones. The specific peptides induced by the digestion are then analyzed by LC-MS/MS. The use of this previous method provides sensitivity and reproducibility necessary for quantification of specific peptides. Results: Almost 100% of the peptide sequence is characterized and 200 peptides are identified by peptide mapping method. From BLAST, 7 peptides have been yet isolated as potentially candidates. We have a strong hypothesis about the complementary determinant receptor (CDR) location on peptides from trastuzumab trypsination. Conclusions and Perspectives: At this point, we are about to confirm experimentally the peptide candidates. The final chosen peptides will be used for LC-MS/MS quantification. The next steps of the study are to develop an extraction method using immunocapture and an optimized quantification method in plasma using UPLC-MS/MS method.
118/381
[P32] Les polyphnols des vins ross : MSn et MRM prliminaires une tude large chelle
Marine Lambert1, Arnaud Verbaere1, Emmanuelle Meudec1, Jrmie Wirth2, Grard Mazerolles1, Gilles Masson3, Vronique Cheynier 1, Nicolas Sommerer1
1
INRA, UMR1083 SPO, Sciences Pour lnologie, Plate-Forme danalyse des polyphnols, 2 place Viala, 34060 Montpellier
2
INRA, UMR1083 SPO, Sciences Pour lnologie, Equipe Polyphnols et Intractions, 2 place Viala, 34060 Montpellier
3
Centre de Recherche et d'Exprimentation sur le Vin Ros, 70 avenue Wilson , 83550 VIDAUBAN
Contact: Marine Lambert (lambertm@supagro.inra.fr)
Keywords: Separative analysis methods, Proteins, peptides and small molecules quantification Les polyphnols, mtabolites secondaires des vgtaux, forment une famille chimiquement trs htrogne et complexe. Issus de fruits, vgtaux, aliments ou produits transforms, ils trouvent leurs applications dans des domaines varis, tels que lindustrie alimentaire, cosmtique ou pharmaceutique, en raison des proprits anti-oxydantes quon leur attribue. Contrairement aux vins rouges, les vins ross contiennent une faible quantit de polyphnols qui jouent toutefois un rle important dans la perception sensorielle (couleur par exemple) du produit fini. Le couplage de la chromatographie ultra-haute performance (UHPLC) la spectromtrie de masse de type triple quadripolaire (ESI-QqQ-MS) permet dallier rapidit, sensibilit et spcificit, et ainsi de classer de grandes sries dchantillons. En effet, le mode de dtection MRM (Multiple Reaction Monitoring) permet de dtecter et doser slectivement des molcules cibles pralablement identifies partir dun ou plusieurs fragments, les donnes gnres permettant ensuite lanalyse statistique et chimiomtrique, dans le cadre dapproches mtabolomiques. Nous prsentons ici la mthode danalyse rapide par MRM des polyphnols contenus dans des vins ross en provenance de diffrentes rgions du monde que nous avons mise au point, pralable ltude trs large chelle (plusieurs centaines de vins du monde). Elle permet de suivre lvolution et de quantifier en une seule injection une centaine de composs polyphnoliques diffrents (anthocyanes, acides phnols, flavonols, flavanols, stilbnes). A terme, les donnes obtenues doivent permettre dlaborer une typologie des vins analyss en corrlant leur composition polyphnolique leur origine gographique, leur cpage, leur couleur, ou de dceler dventuelles fraudes (obtention de vin ros par mlange de vins rouge et blanc notamment). Link: http://www.montpellier.inra.fr/spo/structures_collectives/plate_forme_polyphenols
119/381
[P33] Development of original cross-linkers specifically designed for mass spectrometry analysis
Christian Malosse1, Jingjing Zhao1, Ashley C. Gudzinski1, Guillaume van der Rest 1, Julia Chamot-Rooke1, Sbastien Dautrey2, Anthony Romieu2, Pierre-Yves Renard2
1 2
Laboratoire des Mcanismes Ractionnels, Ecole Polytechnique, 91128 Palaiseau COBRA-CNRS UMR 6014 & FR 3038, rue Lucien Tesnire, 76131 Mont-Saint-Aignan
Contact: Guillaume van der Rest (gvdr@dcmr.polytechnique.fr)
Keywords: Signaling, interactomics and post-translational modifications, High mass and high resolution mass Spectrometry In the variety of approaches to assess ternary and quaternary structure of proteins and protein complexes by mass spectrometry, chemical cross-linking could provide a unique means to obtain direct distance measurements at the molecular level. While the experimental procedure for chemically cross-linking proteins is now well established, there are several analytical challenges that remain in the identification of the cross-linked peptides by MS. A main difficulty is the differentiation of cross-linked peptides from unmodified ones in the extremely complex peptides mixtures that result from digestion of cross-linked proteins. Another difficulty is the ability to identify the specific location of the cross-link using tandem MS. Thus, we have started the development of new chemical cross-linkers giving both a specific signature in the MS (or MS/MS) spectra as well as high sequence coverage in tandem MS experiments. The approach that we have chosen is based on click chemistry. Our new cross-linkers are trifunctional: they bear two reactive groups designed to react with various protein side-chains, whereas the third consists of an alkyne function. This latter will be used for click-chemistry addition of a tag on an azide containing compound (Copper I catalyzed cycloaddition), which will provide a specific marker for mass spectrometry analysis. Two types of tripod cores have served as building blocks: a rigid core (phenyl group) and a soft lysine-like core. Reaction on peptide side-chains can be introduced as two N-hydroxy-succinimide (NHS) activated carbamate groups, or as one NHS activated carbamate and one benzophenone group for photoactivated crosslinking. Cross-linker length can be varied by repetition of (CH2-CH2-O) motives, which will also help solubilizing the tripod. Based on this versatile synthesis strategy, a first series of 6 cross-linkers were synthesized and assayed on a peptide (Substance P) and a small protein (myoglobin). Experimental parameters were optimized to maximize derivatization efficiencies and minimize cross-linker hydrolysis. Among the parameters that were assayed were temperature, reaction time, buffer pH and composition. One interesting observation is that our cross-linkers lead to less dead-end products than the commercial ones. For mass spectrometry analysis, the procedure was optimized to provide efficient detection of the labeled peptides, through the use of a permanent charge added by click-chemistry. Link: http://www.dcmr.polytechnique.fr/spip/spip.php?rubrique33
120/381
[P34] NwCompare et AutoCompare, deux outils pour l'analyse de donnes protomique et transcriptomique.
Frdric PONT1, Jean Jacques FOURNIE2
1
Plateau technique Interactions et Profils d'expression Protiques. INSERM UMR1037-Cancer Research Center of Toulouse, UDEAR. Hpital PURPAN. TSA 40031, 31059 TOULOUSE cedex 09
2
INSERM UMR1037-Cancer Research Center of Toulouse. ERL 5294 CNRS, BP3028, CHU Purpan, 31300 Toulouse Contact: Frdric PONT (frederic.pont@inserm.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics La protomique, la transcriptomique et la mtaobolomique produisent des listes de protines, de gnes ou des noms de mtabolites. Il est souvent trs utile de pouvoir comparer rapidement et facilement ces listes afin de dterminer si des protines, des mtabolites ou des gnes sont spcifiques certains chantillons. NwCompare (*) est un petit logiciel, trs simple d'utilisation, qui permet la comparaison d'un nombre quasi illimit de listes au format texte. Il est donc possible avec nwCompare, de comparer des listes de noms de protines, de gnes, de mtabolites en quelques secondes sans avoir recours une syntaxe complexe et sans avoir besoin d'un serveur ou d'un accs internet. AutoCompare (**) va comparer automatiquement une liste de donne avec des listes de rfrences et calcule la probabilit que la concordance des listes soit due au hasard. Nous avons collect et gnr environ 5500 listes de rfrences, contenant les noms de gnes impliqus dans des fonctions cellulaires, des mtabolismes, des voies de signalisation etc... Avec AutoCompare, il est donc possible d'obtenir trs facilement des informations sur la signification biologique d'une liste de gnes. Comme nwCompare, AutoCompare ncessite peu de puissance et sa prise en main est immdiate. Ces logiciels sont libres et gratuits. Rfrences : (*) Pont F, Fourni JJ. Proteomics. 2010 Mar;10(5):1091-4. Sorting protein lists with nwCompare: a simple and fast algorithm for n-way comparison of proteomic data files. (**) soumis pour publication.
121/381
[P35] Dtection desters de strodes dans le sang par UPLC-MS/MS Une stratgie analytique permettant de rvler certaines pratiques anabolisantes en levage
Zied KAABIA 1, Gaud DERVILLY-PINEL 1, Philippe PLOU 2, Yves BONNAIRE 2, Bruno LE BIZEC 1
1
Laboratoire dtude des Rsidus et Contaminants dans les Aliments (LABERCA), Atlanpole - La Chantrerie, BP 40706, Nantes, F-44307, France, 44307 Nantes
2
Laboratoire des Courses Hippiques (LCH), 15 Rue de Paradis, 91370 Verrires-le-Buisson, France
Contact: Zied KAABIA (zied.kaabia@oniris-nantes.fr)
Keywords: Separative analysis methods La lutte contre le dopage est un sujet en perptuelle volution pour lequel ces dernires dcennies ont vu lessor de techniques de pointe bases principalement sur la spectromtrie de masse, permettant des avances considrables dans la dtection de substances dopantes chez les athltes (humains ou animaux) ou les animaux de production. Si le systme de contrle dans son ensemble nest pas remis en cause, force est de constater quil se heurte aujourdhui certaines limites parmi lesquelles le cas des hormones strodes naturelles qui savre particulirement dlicat traiter. Il semble en effet que certaines substances anabolisantes considres jusqualors comme strictement xnobiotiques puissent tre retrouves, dans certaines conditions, dans les fluides biologiques danimaux nayant pas reus de traitement anabolisant. Si diverses hypothses expliquant ce phnomne sont avances, avec parmi elles une modification de lalimentation, des pratiques de courses diffrentes (dferrage, frquence des courses, saisonnalit, ), une influence de la saisonnalit, ou encore des conditions physiologiques particulires (gestation, blessures, ), il nexiste lheure actuelle aucun critre analytique permettant de statuer quant la conformit de lchantillon face une telle situation. Face ces constats rcents, il apparat en effet primordial de trouver un ou plusieurs critres autorisant la discrimination entre des situations de fraude avec certains strodes et des conditions naturelles dexcrtion urinaire. En particulier, le dveloppement dune mthode autorisant la recherche desters de strodes, composs signant sans ambigut une administration exogne, dans les fluides biologiques (urine / sang) afin dautoriser la mise en vidence directe des composs administrs est une des stratgies analytique privilgie par le LABERCA et le LCH dans le cadre d un programme de recherche collaboratif. Lobjectif de ce travail est de mettre en uvre une mthode analytique rapide pouvant dtecter un large panel desters de strodes ltat de trace (de lordre de la dizaine de ppt) dans le srum/plasma de bovins et dquins. La prparation dchantillon implique un passage de lchantillon aux ultrasons suivie dune tape dextraction en phase solide (SPE). Lidentification des esters de strodes est ralise par UPLC-MSMS sur un instrument de dernire gnration (Xvo TQS, Waters) permettant datteindre des niveaux de sensibilit de la dtection compatibles avec les taux circulants attendus. Lanalyse de srum bovins collects aprs administration de teststrone-propionate ont permis dprouver la mthode et dresser le profil cintique de lester de quelques heures (centaine de ppt) jusqu plusieurs jours aprs son administration.
122/381
[P36] Quantification des Bisphnol-A halogns par HPLC-MS/MS

Emilien JAMIN1, Hanna KULYK1, Isabelle JOUANIN 1, Laurent DEBRAUWER1
1
INRA, UMR1331 TOXALIM, plateforme MetaToul, 180 Chemin de Tournefeuille, F31027 Toulouse
Contact: Emilien JAMIN (jamin@toulouse.inra.fr)
Keywords: Proteins, peptides and small molecules quantification Si le bisphenol-A (BPA) est un perturbateur endocrinien contaminant de lenvironnement bien connu, ses drivs broms et chlors prsentent galement une problmatique toxicologique, mais sont beaucoup moins recherchs. Le ttrabromobisphnol-A (TBBPA) est un retardateur de flamme produit industriellement en grande quantit. Il conduit la formation de produits de dgradation correspondant au tri, di et monobromobisphenol-A (tBBPA, dBBPA et mBBPA). Le ttrachlorobisphenol-A (TCBPA) est quant lui produit en plus faibles quantits en tant que retardateur de flamme, mais peut tre gnr directement par simple chloration du bisphenolA (1), tout comme ses analogues tri, di et monochlorobisphenol-A (tCBPA, dCBPA et mCBPA). Lexposition quotidienne de lHomme ces composs perturbateurs endocriniens est avre et leur prsence dans le tissu adipeux humain a dj t montre (2). Dans ce contexte, nous prsentons dans ce travail le dveloppement dune mthode de quantification de ces analogues halogns du bisphenol-A par HPLC-MS/MS dans des chantillons biologiques, et en particulier dans des chantillons de lait. Ainsi, le BPA, le mBBPA, le dBBPA, le tBBPA, le TBBPA, le mCBPA, le dCBPA, le tCBPA et le TCBPA, ont t quantifis par calibration interne en utilisant ces substances marques par des isotopes stables (carbone 13). Lensemble de ces composs ont t synthtiss au laboratoire partir de BPA et de [13C]-BPA. Les analyses ont t ralises par un couplage HPLC-MS/MS constitu dun systme Accela 600 (Thermo Scientific), dun gradient de phases mobiles mthanol / eau (0,2 mL/min), et dune colonne Modulo Cart QS Uptisphere 3BIO P2 (150 x 2,1 mm), 3 m (Interchim) maintenue 25C. La dtection a t ralise avec un spectromtre de masse TSQ Quantum Discovery MAX (Thermo Scientific) de type triple quadriple oprant en mode MRM avec une source dionisation Electrospray en mode ngatif. La mthode ainsi dveloppe permet de sparer lensemble des composs tudis en 12 min. La dtection est assure sur la base de 3 transitions (2 pour le mBBPA). Les performances de la mthode seront discutes en termes de linarit (suprieure 0,992 pour tous les composs sur une gamme comprise entre 3 et 3000 pg injects), rptabilit (infrieure 14%) et de sensibilit (limites de dtection (S/N>3) comprises entre 0,3 et 3 pg injects selon les composs). Enfin, dans loptique dappliquer notre mthode au dosage des drivs halogns du BPA dans le lait maternel, un protocole dextraction/purification a t dvelopp sur la base dune mthode existante pour le dosage du TBBPA (3), qui utilise diffrentes tapes dextraction liquide/liquide successives, suivies dune purification SPE Si-OH sur cartouches en verre. (1) H. Liu et al., Environ. Sci. Technol. 43 (2009) 7712 (2) M.F. Fernandez et al., Reprod. Toxicol. 24 (2007) 259 (3) R. Cariou et al., J. Chromatogr. A 1100 (2005) 144
123/381
[P37] Spciation de liode en milieu biologique par desorption/ionisation par electrospray (DESI)
Diane Lebeau1, Jean Baptiste Fournier2, Catherine Leblanc2, Pascal Reiller1, Thierry Pourcher3, Denis Doizi1
1 2 3
CEA-Saclay, DEN/DANS/DPC/SECR/LSRM, 91191 Gif-sur-Yvette UMR 7139, CNRS-UPMC, Station Biologique, 29680 Roscoff Unit TIRO, CEA-Universit de Nice, 06107 Nice
Contact: Diane Lebeau (diane.lebeau@cea.fr)
Keywords: Organic and inorganic mass spectrometry Liode est un oligolment prsent dans la plupart des formes de vie volue et essentiel la vie humaine. Il est principalement prsent dans les mers et les produits marins et se retrouve dans une moindre mesure dans les terres et diverses plantes et animaux. Il peut tre relargu dans latmosphre sous diverses formes chimiques inorganiques (I-, I2, IO3-, HIO) ou organiques (CH3I et autres drivs alkyls ou phnoliques). La rpartition de liode entre ses diffrentes formes chimiques conditionne son devenir et son impact sur lenvironnement. Chez les mammifres, un apport quotidien, essentiellement par lalimentation, est ncessaire pour notamment, la synthse des hormones thyrodiennes. Lobjectif de ce travail a consist en la mise au point dune mthode analytique permettant la spciation par spectromtrie de masse de liode dans son environnement biologique. Ainsi deux modles naturels ont pu tre tudis : la surface dalgues brunes Laminaria digitata et le contenu du tractus digestif de souris. Les laminaires sont dimportants accumulateurs diode et des contributeurs majeurs au cycle de liode en milieu marin cotier. Le deuxime systme tudi a pour but de caractriser la biodistribution et le mtabolisme de liode au niveau du tractus digestif. Liode est constitutivement accumul au niveau de lestomac et des glandes salivaires et il est absorb au niveau de lintestin. Une des finalits de ce travail est notamment un meilleur contrle de lexposition des organes en cas de contamination par un isotope radioactif (125,129,131I). La dsorption/ionisation par electrospray (DESI) est lune des dernires techniques analytiques utilises en spectromtrie de masse permettant la dtection de molcules organiques directement dans leur environnement pression atmosphrique. Cette mthode a donc t choisie afin de caractriser liode sur les deux modles dtude choisis. Cette technique dionisation douce permet, en effet, lanalyse de la spciation dun grand nombre despces organiques ou inorganiques. Il a ainsi t possible de dtecter les diverses formes de liode sur ces deux modles tudis. Il a t montr que la dtection des ions I- et IO3- sur des coupes de colon enrichis en iode par addition de solutions de NaI et NaIO3 par DESI tait possible. Ainsi, la spciation de liode a t conserve, les ions I- (m/z 126,9) et IO3- (174,9) ayant t respectivement dtects. Loptimisation des conditions exprimentales (choix du solvant, paramtres gomtriques de la source, pression de gaz, tension du capillaire, etc) a ensuite permis datteindre une limite de dtection de lordre de 0,2 nmol/mm. La concentration diode estime tant de lordre du M, cette limite de dtection obtenue est donc parfaitement compatible avec nos objectifs. Une tude de la distribution de ces diffrentes espces organiques et inorganiques de liode sur la surface des modles est en cours.
124/381
[P38] Avantages des couplages de la chromatographie de partage centrifuge avec la chromatographie liquide et la spectromtrie de masse pour la purification de composs dans des extraits de plante
Emilie Destandau1, Thomas Michel1, Alix Toribio2, Claire Elfakir1
1 2
Institut de Chimie Organique et Analytique, rue de chartres, 45067 orleans phycosource, 13 boulevard de l'Hautil, 95000 Cergy pontoise
Contact: Emilie Destandau (emilie.destandau@univ-orleans.fr)
Keywords: Separative analysis methods, Organic and inorganic mass spectrometry, Instrumentation La Chromatographie de Partage Centrifuge (CPC) utilise un mlange liquide biphasique pour sparer ou purifier des molcules lchelle semi-prparative. Grce son absence de support solide et sa grande capacit dinjection, la CPC savre tre une technique de choix pour la sparation ou la purification de molcules naturelles partir dextraits de plantes. Lors de la purification des molcules dintrt la CPC est gnralement couple un dtecteur UV qui permet dobtenir le chromatogramme CPC de lextrait. Dans ce cas, les informations structurales sont faibles et les composs isols sont analyss off-line par spectromtrie de masse (SM) afin de les identifier. Le dveloppement du couplage CPC-MS permet davoir immdiatement lors de la purification, un accs direct des informations structurales sur les molcules dintrt ; ainsi le suivi de purification est facilit et plus rapide. Lorsque lextrait de plante est complexe et que les molcules ne peuvent pas tre purifies une une, la CPC peut tre utilise dans le but de fractionner cet extrait complexe ce qui permet dobtenir une empreinte plus exhaustive de lextrait et de faciliter lidentification ultrieure des composs dintrt par LC-MS. En permettant dassocier en simultan lempreinte chromatographique des diffrentes fractions et les spectres de masse des diffrents constituants, le dveloppement innovant du couplage CPC-LC-MS favorise et facilite le guidage en ligne du fractionnement. Les deux couplages de la CPC directement avec la SM ou avec la LC-MS sont dlicats de part, le changement dchelle prparative pour la CPC et analytique pour la LC et la MS, et de part le risque de non miscibilit des solvants utiliss. Une fois dvelopps et optimiss, ces couplages ont t respectivement appliqus la purification de xanthones dans un extrait de mangoustan et au fractionnement de flavonodes dans un extrait de baies dargousier. Un rel gain sur la qualit du fractionnement, sur lidentification structurale et sur la dure totale des expriences a t observ.
125/381
[P39] Study of ESBL producing- and beta lactam-sensitive E.coli using MALDI-TOFMS and hybrid MALDI-QIT-TOF tandem mass spectrometry
Omar BELGACEM1, Emmanuel WEY2, Indran BALAKRISHNAN2, Shervanthi HOMER-VANNIASINKAM3, Valery EDWARDS-JONES3, Pranav SOMAIYA3
1 2 3
SHIMADZU KRATOS, Trafford Wharf Road - Wharfside, M17 1GP MANCHESTER (UK) ROYAL FREE HOSPITAL, , LONDON (UK) NPIMR-UCL, , LONDON (UK)
Contact: Omar BELGACEM (omar.belgacem@kratos.co.uk)
Keywords: Clinical proteomics Introduction The emergence of multi-drug resistant (MDR) enterobacteriaceae that can produce Extended Spectrum -lactamses (ESBLs) is currently a major worldwide concern. ESBLs are a group of enzymes that are transfered via plasmids in gram negative bacteria. They confer reistance to a wide spectrum of beta-lactam antibiotics currently used as 1st line empirical therapy in the management of gram negative bacterial infections. E.coli is the most frequent gram-negative rod isolated from blood cultures in clinical settings. Microbial biotyping is generally obtained using fingerprinting methods. Here, we deviate from MS-only pattern recognition methods and use tandem mass spectrometry to find biomarkers in ESBL producers. Polypeptides from ESBL producing E.coli samples were desorbed directly from the MALDI plate using hybrid MALDI-QITTOF instrumentation. Method Two groups of organisms beta lactam sensitive E.coli and an ESBL-producing (ESBLp) E.coli were grown and analysed by MALDI-TOF and MALDI-QIT-TOF. Preliminary Data Linear MS of the two organisms showed consistent reproducibility in replicate of linear mode spectra obtained in the 2-20 kDa mass range. There were significant differences between beta lactam sensitive and ESBLp E.coli. The difference was such that no identifications could be obtained with the ESBLp E.Coli. Two peaks at 2341.3 Da and 3790.0 Da detected in the ESBLp E.coli spectra exhibited unusually high intensity relative to other peaks. When subjected to MS2 analysis using the reflectron mode of operation, we obtained no workable tandem MS. This was explained by the unusual crystallization process observed for ESBLp E.Coli which led ultimately to poor ionization efficiency. In order to avoid adverse effects of uneven sample surfaces, we decided to use a hybrid MALDI-QIT-TOF instrument where the ionization source is decoupled from the TOF analyser typical for such instrument configuration. CID experiments performed on several peaks allowed us to detect two main components that exhibited isotopic resolution across the whole mass range. After performing a Mascot search we identified two peptides (2341.3 Da and 3790.0 Da) originating from the same acid shock protein. The fragmentation pattern of these amino acid sequences were exhibiting b- and y-type ion series and hence led to identifications with high confidence. This protein is known to be found in the periplasm and our results indicate that such proteins seem to be easily detected in the ESBLp sample. The presence of several lysine residues in the detected peptides could also explain its high proton affinity and this may explain the lack of detection of other protein signals in this sample. Future work will consist of explaining the presence of this protein in the ESBLp E.Coli sample as well as investigate if such methods can be used for different organisms. Link: www.shimadzu.com
126/381
[P40] Evidence for the involvment of anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation by global approaches.
Patricia Costaglioli 1, Christophe Barthe1, Stphane Claverol2, Michel Perrot2, Marc Crouzet1, Marc Bonneu2, Bertrand Garbay1, Sbastien Vilain2
1
Laboratoire de Biotechnologie des Protines Recombinantes Vise Sant, 146 rue Lo Saignat, 33076 BORDEAUX Cedex
2
Ple Protomique, 146 rue Lo Saignat, 33076 BORDEAUX Cedex
Contact: Sbastien Vilain (sebastien.vilain@u-bordeaux2.fr)
Keywords: Proteins, peptides and small molecules quantification Pseudomonas aeruginosa is a genetically complex bacterium which can adapt to its environment andswitchbetween a free-living or biofilm lifestyle. This versatility enables P. aeruginosa to thrive in many different environments and contributes to its success as a human pathogen. Biofilms are complex bacterial communities found attached to biotic or abiotic surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA and proteins produced by the bacteria. In this work we investigated the genome expression of sessile cells present in biofilms developed on glass wool substratum, which can concentrate extracellular DNA. By using microarrays and mass spectrometry associated with iTRAQ labelling, transcriptome and proteome of sessile cells were explored and compared with those of two kind of planktonic cell cultures , one in exponential growth phase and the other one in stationary phase. A statistical study by Principal Component Analysis revealed the existence of a specific genome expression during biofilm formation and the expression of a cluster of mRNAs and proteins was specifically modified when compared to planktonic cells. We observed an overexpression of Mg2+ regulon and of genes involved in the production of the low-affinity siderophore pyochelin system. The Rlh and PQS systems were also upregulated in biofilms when compared to exponentially growing cells. The sessile cells obtained in our experimental model exhibited several features with P. aeruginosa isolated from the lungs of cystic fibrosis patients. Our study also highlighted the overexpression of genes controlling the anthranilate degradation pathway in the sessile cells grown for 24 hours on glass wool. In addition, we did not observe any significant induction of the metabolic pathway which uses anthranilate for PQS production. These results suggest that anthranilate was mainly used for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation by P. aeruginosa. The phenotype analyses confirmed that biofilm formation was partially dependent on the activity of the anthranilate degradation pathway. This work revealed a new feature concerning the anthranilate use in P. aeruginosa sessile cells.
127/381
[P41] Glycosylation Profiling of Therapeutically Active Glycoproteins: Identification of Glycans in Recombinant Factor IX using Directed MALDI-QIT-TOF-MSn and a Glycan Database
Matthew OPENSHAW1, Daniel SPENCER2, Louise ROYLE2, Katharina POCK3, Omar BELGACEM1
1 2 3
SHIMADZU KRATOS, Wharfside, Trafford Wharf Road , M17 1GP Manchester (UK) LUDGER Ltd, , Oxford (UK) OCTAPHARMA PHARMAZEUTIKA PRODUKTIONS, , Vienna (Austria)
Contact: (omar.belgacem@kratos.co.uk)
Keywords: Signaling, interactomics and post-translational modifications

Introduction High levels of quality control are required for the characterisation of recombinant biopharmaceuticals to ensure batch-to-batch reproducibility, safety and efficacy. Post-translational processing of recombinant proteins must be carefully characterised and methods must be developed for the profiling of such glycosylation patterns in the products of biomanufacturing. Due to the branched nature of glycans, the unambiguous identification of glycan structures is not trivial. Here, we use a software-directed multistage MALDI mass spectrometric approach which guides the user through the structural analysis of glycans with the aim of identifying a single structure contained in a glycan database, without the need for manual interpretation of MSn data. Preliminary Data Methods for the release, capture, PA-derivatisation & purification of free glycans have been optimised for subsequent MALDI analysis. Released glycans were captured using a combination of C18 and porous graphite carbon (PGC). Reductive amination (PA-labelling) was performed using 2-aminopyridine and 2-picoline borane. Optimisation of the labelling conditions was performed in an attempt to produce the highest yield of labelled glycans whilst minimising non-specific modification of the glycans. We have compared previously published methods for subsequent purification of PA-labelled glycans (solid phase extraction, gel filtration (Sepharose-based method), acetone precipitation, porous graphite carbon) with the aim of providing a clean sample suitable for direct MALDI analysis without the need for HPLC purification. Without effective sample clean-up, excess reagents from the PA-labelling procedure can reduce sensitivity in subsequent MALDI analysis. We applied the optimised sample preparation protocols to the analysis of glycans released from Fetuin (test protein) and recombinant Factor IX (BeneFix (Wyeth Europa, Maidenhead (UK))) to demonstrate the application of the described workflow for the quality control of biotherapeutics. Using this approach, we have identified several glycans from fetuin and have detected several glycans released from the blood coagulation factor IX sample. We will apply the optimised sample preparation protocols and will attempt to identify the factor IX N-glycans using this workflow. Identification of sialylated glycans is more challenging and work is on-going to apply the described approach for the identification of these sialic acid-containing glycans. While QC of such biotherapeutics is typically performed using HPLC-based methods, the MALDI-based identification method could significantly reduce analysis time in cases where differences in glycosylation pattern (relative to a reference batch) are observed. Using the software-directed MSn approach, glycan structures can be identified without the need for interpretation of MSn data. Link: www.shimadzu.com
128/381
[P42] Identification and quantification of Serine/Threonine/Tyrosine phosphorylated proteins of wild type and mutants of Listeria monocytogenes
Sandeep MISRA1, Eliane Milohanic2, Josef Deutscher2, Francine Ak2, Ivan Mijakovic3, Vronique Monnet1, Cline Henry1
1
Plateforme d'Analyse Protomique Paris Sud Ouest, UMR 1319 MICALIS, Domaine de Vilvert, 78352 Jouy en Josas
2 3
Physiologie et Gntiques des Bactries, UMR 1319 MICALIS, Domaine de Vilvert, 78352 Jouy en Josas INRA-AgroParisTech, Domaine de Vilvert, 78352 Jouy en Josas
Contact: Cline Henry (celine.henry@jouy.inra.fr)
Keywords: Proteins, peptides and small molecules quantification , High mass and high resolution mass Spectrometry Listeria monocytogenes is a gram positive, food borne pathogen that is responsible for listeriosis. The infection with L. monocytogenes causes abortion and leads to septicemia and other complications mainly in newborn babies, immunocompromised adults or senior citizens. In this bacterium, two interconnected events, carbohydrate utilization and protein phosphorylation are known to be involved in virulence. The importance of phosphoproteins in bacteria was only recently recognized and is still not well understood. Phosphoproteomes of several bacteria have been deciphered. These studies suggest that Ser/Thr/Tyr phosphorylation is involved in a diverse range of cellular regulatory networks and controls numerous physiological processes, including virulence in bacteria. The objective of the present work is to identify and quantify L. monocytogenes, proteins phosphorylated at Ser/Thr/Tyr residues in order to investigate their role in the regulation of virulence factors, especially via sugar utilization. We are working on a gel free approach to determine the phosphoproteome of L. monocytogenes. After digestion of total proteins with trypsin and endopeptidase Lys-C, the resulting phosphopeptides are enriched in two steps: first by cation exchange chromatography followed by enrichment on titanium beads. The samples are run on an LTQ Orbitrap mass spectrometer and phosphopeptides are identified by using different algorithms: Mascot in conjunction with Maxquant, Sequest and X!Tandem. All the sites of phosphorylation are validated by Ascore. The quantitative phosphoproteome of a prpC (stp) mutant, with innactivated S/T-specific P-protein phosphatase, are compared to the wild-type strain using SILAC. Quantification is realized via freely available softwares QUIL and MassChroQ*. *http://pappso.inra.fr/bioinfo/
129/381
[P43] EasyMSI: a competitive software tool for the interpretation of Mass Spectrometry Imaging datasets
Jean-Pierre BOTH1, Vincent PICAUD1, Marie-France ROBBE1, Isabelle ESPAGNON1
1
CEA Saclay, LIST, Laboratoire d'Outils d'Analyses de Donnes, Btiment 516, 91191 Gif sur Yvette cedex
Contact: Jean-Pierre BOTH (jean-pierre.both@cea.fr)
Keywords: Imaging mass spectrometry EasyMSI is a software tool for spatial and spectral visualization and processing of mass spectrometry imaging, with several modules providing user assistance for the interpretation of data. It has been developed by CEA-LIST during the Computis European project (2006-2009) and the Masda-Eye ANR project (2010-2012). EasyMSI can read MALDI and SIMS datasets in Analyze format (Applied Biosystems), GRD format (IonTof) and the imzML standard format for mass spectrometry imaging based on mzML. EasyMSI provides basic functionalities for data display and exploration (spectrum and image display, peak and pixel picking, zooming on spectra and images, ROI selection), and some more specialized treatments such as denoising spectra or structure analysis. EasyMSI offers the advantage to display MALDI data without binning. The user can choose to bin or not SIMS data, and the associated binning rate. As assistance to the interpretation of data, EasyMSI computes several indicators. The relative variance spectrum enhances peaks that have a highly contrasted space distribution. The Moran index is an autocorrelation indicator adapted to detect thin local structures such as membranes, in images. The correlation spectrum associated to a given m/z brings out correlated m/z, often co-localised or complementary with the given m/z. EasyMSI is able to extract the peak list of the total spectrum, with parameterized criteria of extraction, to best adapt to the dataset peak shapes versus noise. Peak list can be used for molecule identification through the interrogation of biological databases. EasyMSI also includes clustering tools to perform spatial (i.e. pixel-based) classification or spectral (i.e. m/z-based) classification on peak lists or binned data. The K-means clustering is one of the simplest and fastest classification methods. The time for running a K-means clustering usually lasts only some seconds. The hierarchical clustering enables to perform clustering inside a zone defined by a preceding clustering. The diffusion map method consists in reducing dimensionality by embedding data in a space in which data are more easily synthesized, and to do a clustering analysis in the reduced data.
130/381
[P44] Comparaison Triple-Quadriple versus hybride quadriple/temps-de-vol pour la quantification de peptides prototypiques de cytochromes P450
Ahmad AL ALI1, David Touboul2, Alain Brunelle2, Olivier Laprvote3, Philippe Beaune1
1
Bases Molculaires de la rponse aux xnobiotiques, INSERM UMR-S775, Centre Universitaire des SaintsPres, 45 rue des Saint Pres, 75270 Paris Cedex 06 Paris, France
2
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex Gif-sur-Yvette, France
3
Chimie Toxicologie Analytique et Cellulaire, EA4463, Facult des Sciences Pharmaceutiques et Biologiques, Universit Paris Descartes, 4 avenue de lObservatoire, 75006 Paris Paris, France Contact: Ahmad AL ALI (alali@icsn.cnrs-gif.fr)
Keywords: Proteins, peptides and small molecules quantification Les cytochromes P450 (CYP) sont des enzymes essentielles dans le mtabolisme des xnobiotiques. Ils participent de faon centrale llimination des xnobiotiques et dans certains cas la production de mtabolites toxiques et/ou ractifs. Il est trs important dtre capable de les doser individuellement avec prcision, compte tenu de leur spcificit en terme de ractivit, ce qui est rendu dlicat par leurs similitudes de squences. Les CYPs sont en gnral quantifis par amplification de gnes (PCR) ou immuno-marquage mais ces mthodes donnent soit une information indirecte (quantit dANR messager), soit une information lacunaire (difficult de produire des anticorps spcifiques pour les CYP minoritaires). La spectromtrie de masse, permettant danalyser de trs faibles quantits de protines en mlange, peut tre considre comme une mthode de choix pour lidentification et la quantification de CYP. Trois CYPs (1A2, 2D6, 2J2) ont t choisis comme modles. Aprs digestion trypsique des protines sur-exprimes dans des baculosomes, 7 peptides prototypiques ont t identifis puis synthtiss. Afin dvaluer la technique la plus sensible et robuste de dosage par spectromtrie de masse, deux instruments (triple quadriple et hybride quadriple/temps-de-vol avec cellule de mobilit ionique) utilisant diffrents modes danalyses (SIM, pseudo-SRM, MRM) et coupls une chane UPLC ont t valus. En mode SIM, les deux appareils ont montr des performances identiques. La mobilit ionique ne permet pas une quantification des peptides mais peut aider leur identification. Finalement le mode MRM du triple quadriple savre moins sensible mais plus spcifique que le mode SIM. Afin de finaliser cette tude, les peptides prototypiques marqus 13C et 15N vont tre synthtiss et les diffrents modes danalyse seront compars sur des chantillons biologiques complexes (biopsies de foie et de cerveau humains).
131/381
[P45] Nucleotide cycle in the archeal aIF2 system probed by native and denaturing mass spectrometry.
Christian Malosse1, Emmanuelle Schmitt2, Yves Mechulam2, Michel Panvert2, Guillaume van der Rest1
1 2
Laboratoire des mcanismes ractionnels DCMR, Ecole polytechnique route de saclay, 91128 palaiseau Laboratoire de biochimie, Ecole polytechnique route de saclay, 91128 palaiseau
Contact: Christian Malosse (malosse@dcmr.polytechnique.fr)
Keywords: Systems biology, High mass and high resolution mass Spectrometry In eukaryotes, a 43S complex, comprising a ribosomal 40S subunit, eukaryotic initiation factors (eIF), 1, 1A, 3, 5 and eIF2:GTP:Met-ARNtiMet binds to the 5-capped end of mRNA with the help of eIF4s and scans downstream to the initiation codon to form a 48S complex. Regulation of the nucleotide cycle of e/aIF2 is crucial for the accuracy of the initiation of translation. In archaea, orthologs of eukaryotic subunits are presents and the corresponding heterotrimeric factor was therefore named aIF2. However, archaea have no equivalent of the catalytic subunit of eIF2B (eIF2B and of eIF5. Therefore, GTP hydrolysis on aIF2 is likely to occur without GAP assistance, and the recycling of aIF2-GDP into aIF2-GTP is thought to be spontaneous. Nevertheless, until now, no measurement of the kinetic or equilibrium association constants for GTP and GDP binding to aIF2 are available. Therefore, new methods allowing such measurements are required to understand the nucleotide cycle on archaeal aIF2 and to give new insights into the specificity of the initiation of translation in archaea. Non-covalent and dissociative mass spectrometry approaches have been carried out using QTOF nano-ESI experiments on the Sulfolobus solfataricus aIF2 complex incubated with various nucleotides. Native mass spectrometry measurements were conducted after Bio-Spin column desalting of the initial protein solution. Measurement of the purified native complex led to the observation of an intact complex (charge states 19-23+, 93 kDa) containing all three subunits, as well as a Zn2+ cation and a GDP nucleotide. The latter was unexpected, as the solution did not contain any additional GDP, which should have shifted the equilibrium towards complete GDP loss. Furthermore, after incubation in GDP, GDP-NP, or GTP, one observes formation of complexes bearing two nucleotides, indicating the presence of a second, weaker binding site. Gas-phase dissociation of the native complex gave insights as to the general topology of the complex, and the subunits on which the cofactors (Zn2+, nucleotides) are bound. Denaturing the complex gives access to the individual elements of the complex, and interestingly allows measurement of the incorporation of GDP vs GTP (or GDP-NP), which is not possible on the intact complex. Combining native and denaturing approaches on the same samples leads to the determination of both kinetic and thermodynamic parameters for the aIF2 nucleotide binding.
132/381
[P46] Photofragmentation and action spectroscopy of gas phase peptide and protein polyanions. From near-UV to vacuum UV.
Claire Brunet1, Rodolphe Antoine1, Philippe Dugourd1, Francis Canon2, Alexandre Giuliani2, Laurent Nahon2
1
Laboratoire de Spectromtrie Ionique et Molculaire, Bat. A. Kastler, 43 bd du 11 novembre, 69622 Villeurbanne Cedex
2 3
SOLEIL, l'Orme des Merisiers, St Aubin, 91192 Gif sur Yvette Cedex Cepia, Institut National de la Recherche Agronomique (INRA), BP 71627, 44316 Nantes
Contact: Claire Brunet (cbrunet@lasim.univ-lyon1.fr)
Keywords: Organic and inorganic mass spectrometry Photon activation methods are emerging techniques[1, 2] complementary to collision or electron based methods for structural characterization of peptides and proteins. Especially in the UV range, the photon induces an electronic excitation of molecular ions. After excitation, direct dissociation in excited states competes with internal conversion to the electronic ground state and with radiative de-excitation. Therefore, new fragmentation channels may be opened. For peptide and protein polyanions containing natural aromatic amino-acids, electron photodetachment is the main relaxation channel observed after UV excitation[3]. This results in a radical species, which can be isolated and further activated by collisions (activated-EPD)[4]. Experiments on peptides and proteins will be described in this presentation. The synchrotron radiation allows us to work in the vacuum UV (under 200 nm), where electronic excitations are originating from absorptions of different natures and locations in polypeptides and may open new pathways for electron loss and/or fast dissociation products. Results on caerulein model peptide will be given as example in this presentation. Besides activated-EPD experiments, electron photodetachment yield as a function of photon energy can be used to record optical spectra of peptide and protein ions. Action spectra for both non-radical and radical caerulein ions and protein polyanions will be presented[5].
1. Ly, T. and Julian, R.R., Ultraviolet Photodissociation: Developments towards Applications for Mass-Spectrometry-Based Proteomics. Ang. Chem. Int. Ed., 2009. 48, p. 7130-7137. 2.Reilly, J.P., Ultraviolet Photofragmentation of Biomolecular Ions. Mass Spectrom. Rev., 2009. 28, p. 425-447. 3.Joly, L., et al., Ultraviolet Spectroscopy of Peptide and Protein Polyanions in Vacuo: Signature of the Ionization State of Tyrosine. J. Am. Chem. Soc., 2007. 129, p. 8428-8429. 4.Larraillet, V., et al., Activated-Electron Photodetachment Dissociation for the Structural Characterization of Protein Polyanions. Anal. Chem., 2009. 81, p. 8410-8416. 5.Brunet, C., et al., Vacuum UV Electron Photodetachment Dissociation of Peptide Polyanions. Submitted. Link: http://www-lasim.univ-lyon1.fr/spip.php?rubrique8
133/381
[P47] Etudes de ladsorption des protines de plasma riche en plaquettes sur un biomatriau en titane bioactiv avec du polystyrne sulfonate de sodium
S. Oughlis 1, F. Poirier1, S. Lessim1, S. Changotade1, G. Helary1, F. Bollotte1, J.J. Lataillade2, V. Migonney1, D. Lutomski1
1
UMR CNRS 7244 - CSPBAT LBPS , 74 rue M. Cachin UFR, SMBH Lonard de Vinci, Universit Paris 13 , 93000 Bobigny, France
2
Laboratoire de thrapie cellulaire, CTSA, HIA Percy , 92140 Clamart
Contact: S. Oughlis (oughlis.sophiane@yahoo.fr)
Keywords: Separative analysis methods, Organic and inorganic mass spectrometry Le titane est couramment utilis dans les implants orthopdiques et dentaires pour son excellente rsistance la corrosion et sa biocompatibilit. Malgr cela, 15% des prothses poses chaque anne en France, sont des r-interventions aprs chec de la prothse de premire intention. La principale cause dchec reste des problmes dostointgration avec descellement aseptique par dcohsion de linterface os/implant, due une destruction progressive des tissus biologiques au contact de la prothse (formation dun tissu fibreux). Afin damliorer lostointgration long terme du titane, nous avons dvelopp des protocoles permettant de greffer de manire covalente des groupements chimiques la surface du mtal. Cette modification chimique par polymrisation radicalaire de polymres bioactifs porteurs de groupements ioniques tels que le sulfonate (polystyrne sulfonate de sodium) permet de moduler la fixation des cellules et l'activit cellulaire des ostoblastes. Nanmoins, la fixation biologique de la prothse los est influence par plusieurs facteurs, incluant la nature de la prothse, la chimie de surface mais galement les protines adsorbes la surface. Le plasma riche en plaquettes (PRP) contient des facteurs de croissance connus pour favoriser la rgnration osseuse et pour avoir des applications potentiels en ingnierie tissulaire. Dans le cadre de cette tude, lutilisation combine du titane bioactiv et du PRP a t envisage. Du PRP a t adsorb de faon non covalente sur la surface du titane bioactiv. Pour identifier les protines adsorbes sur la surface de titane, dans un premier temps, les protines ont t dsorbes du biomatriau. Elles ont ensuite t spares par lectrophorse bidimensionnelle avant dtre identifies par spectromtrie de masse par la technique dempreinte peptidique massique. Les proprits de surface montrent une slectivit du titane bioactiv vis--vis de certaines protines du PRP. Dans un second temps, des tudes in vitro ont t ralises afin dtudier le comportement de cellules souches msenchymateuses humaines (CSM) sur du titane bioactiv sur lequel des protines de PRP ont t adsorbes. Link: http://www.univ-paris13.fr/cspbat/accueil.html
134/381
[P48] Structure and dynamics of membrane proteins using hydrogen/deuterium exchange: how to cope with detergents.
Shahid Mehmood1, Martial Rey2, Benjamin Clmenon2, Ludovic Pelosi2, Jean-Michel Jault1, Petr Man3, Eric Forest1
1 2 3
Institut de Biologie Structurale, 41 rue Jules Horowitz, 38027 Grenoble, France Laboratoire de Biologie Grande Echelle, iRTSV, 17 rue des Martyrs, 38054 Grenoble, France Institute of Microbiology, Videnska 1083, 14220 Prague, Rpublique Tchque
Contact: Eric Forest (eric.forest@ibs.fr)
Keywords: Systems biology Membrane proteins represent 30% of the proteins coded by the human genome and 50% of drug targets. Although it is critical to understand the interplay between structure, function and dynamics, elucidation of their structure remains a major challenge due to their hydrophobic nature. HDX MS offers an alternate approach to probe structure and dynamics of membrane proteins even though developments are needed to cope with detergents, necessary for solubilization, but often deleterious to MS. Different transmembrane proteins were extracted with different detergents: dodecyl maltoside (DDM), Triton X100 or C8E4, chosen among several for their capacity to solubilize them and to be eliminated before reaching the ESI source. After deuteration in vitro, or directly in the mitochondria, the membrane proteins were digested with immobilized pepsin or rhizopuspepsin in presence of 1.5 M guanidinium chloride. In the case of DDM the separation column was connected to the electrospray source until 30% acetonitrile and then disconnected to avoid pollution of the source by the detergent. The other detergents were washed with dichloromethane after proteolysis of the deuterated proteins and before elution of the peptides. The ABC transporter BmrA exports multiple drugs out of the cell in vivo, hence conferring multidrug resistance. It is capable of hydrolyzing ATP in a transient closed conformation. After membrane extraction with DDM, HDX was compared for open and closed conformations, giving information on the transport mechanism at the level of the nucleotide binding and intracellular domains. VDAC, a beta-barrel porin from the outer membrane of mitochondria, was extracted with C8E4. HDX showed that the N-terminal part is structured, in agreement with the X-ray structures (but in contrast with NMR observations). The ADP-ATP carrier, in the inner membrane of mitochondria, imports ADP from the intermembrane space and exports ATP from the matrix. After extraction with Triton X100, the comparison of HDX for two different inhibited states of the bovine carrier enabled us to propose a model of the translocation mechanism. After direct deuteration of bovine mitochondria and fast extraction of the carrier the difference of HDX for both inhibited forms was similar to the differences observed in vitro except for the matrix loops. These loops are much less exchangeable in the in situ experiments, indicating probably a stronger interaction with the native membrane. Finally the yeast ADP-ATP carrier was also studied. No X-ray structure has been solved. However HDX results were compared with biochemical and mutagenesis data, giving further evidence that the translocation mechanism involves a conformational change of matrix loop m2. These HDX MS studies on different transmembrane proteins and with different detergents could be extended to gain insight on the structure and dynamics of various membrane proteins.
135/381
[P49] Unusual Peptide fragmentation by C-terminal residue exclusion in ESI and MALDI Tandem Mass Spectrometry
Christine Enjalbal1, Mathieu Dupr1, Sonia Cantel1, Jean Martinez1
1
Institut des Biomolcules Max Mousseron (IBMM), UMR 5247, Universit Montpellier 2, 34095 Montpellier
Contact: Christine Enjalbal (enjalbal@univ-montp2.fr)
Keywords: Organic and inorganic mass spectrometry By screening a dataset of 150 synthetic peptides MS/MS spectra, we found that an unexpected fragmentation was occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTof and Tof/Tof mass analyzer configuration, respectively. The residue positioned at the Cterminal end was lost provided that a basic amino acid, such as arginine and to a lesser extent histidine and lysine, was present in the sequence leading to a new y-type ion corresponding to a shortened peptide. Such a residue exclusion from the sequence gave a fragment ion that was in certain cases the base peak of the MS/MS spectrum. A potent fragmentation mechanism was envisaged within the frame of the mobile proton model. Thus, regarding proteomics studies, any miscleavage from trypsin protein digestion or the use of other proteolytic enzymes (such as Lys-N for instance) will lead to arginine-containing peptides showing such parasite fragmentation from the MH+ precursor ion.
136/381
[P50] Apport de la spectromtrie de masse haute rsolution pour la dtection cible ou non cible de PBDE et de leurs mtabolites par LC-APPI-MS.
Charlotte MARTEAU1, Sylvie CHEVOLLEAU1, Jean Philippe ANTIGNAC3, Bruno LE BIZEC3, Daniel ZALKO2, Laurent DEBRAUWER1
1 2 3
INRA, TOXALIM UMR1331 INRA-INP-UPS, plateforme MetaToul, BP 93173, 31027 Toulouse, France INRA, TOXALIM UMR1331 INRA-INP-UPS, BP 93173, 31027 Toulouse, France ONIRIS, USC 2013 INRA, LABERCA, Atlanple-La Chantrerie, BP 50707, , 44307 Nantes, France
Contact: Laurent DEBRAUWER (laurent.debrauwer@toulouse.inra.fr)
Keywords: Organic and inorganic mass spectrometry, Separative analysis methods Les polybromodiphenylethers (PBDE) constituent lune des principales familles de retardateurs de flamme broms (RFB) utiliss dans lindustrie des plastiques et des polymres. Leur prsence a t mise en vidence dans tous les compartiments environnementaux, et leur devenir dans lenvironnement et chez lanimal constitue une proccupation importante. Cependant, lanalyse de ces composs et de leurs mtabolites reste un dfi analytique important en raison la fois de leur diversit en termes de poids molculaire (pouvant aller de 250 950) et de la complexit des mlanges de congnres natifs ou mtaboliss. Pour ces raisons, bien quelle prsente certaines limitations pour lanalyse des congnres fortement broms ainsi que pour certaines classes de mtabolites, la GC-MS reste lheure la technique la plus utilise pour la dtection des PBDE [1]. Lors de prcdents travaux, nous avons montr que la photo-ionisation pression atmosphrique (APPI) pouvait constituer une mthode performante pour lanalyse des PBDE par LC-MS [2,3], et tudi le comportement de ces composs vis--vis de cette technique dionisation [4]. Nous prsentons ici une tude sur le dveloppement dune mthode LC-APPIMS haute rsolution sur un instrument pige orbital, pour lanalyse de PBDE et de leurs mtabolites par LC-MS. Plusieurs colonnes de type UPLC (greffages C18 ou PFP, 100mm ou 150 mm) ont t testes pour leur capacit sparer des mlanges de PBDE et PBDE hydroxyls pouvant comporter un nombre important disomres de position, et dont la sparation par HPLC reste difficile. Les paramtres dionisation par APPI (nature et % de dopant, dbit de phase mobile, temprature du nbuliseur chauff) ont ensuite t optimiss. Le comportement de diffrents congnres modles de PBDE et PBDE hydroxyls vis--vis de lionisation APPI a t tudi de faon identifier les signaux de travail les plus pertinents. Nos rsultats montrent que la spectromtrie de masse haute rsolution offre une meilleure slectivit de dtection que la MRM tudie en comparaison, avec une sensibilit quivalente. Une application lidentification de mtabolites du BDE-47 forms in vitro sera prsente, montrant que lacquisition en mode spectre complet haute rsolution permet non seulement la dtection de mtabolites cibls mais galement lidentification de nouveaux mtabolites non cibls. [1] A. Vonderheide, Microchem. J. (2009) 92, 49-57. [2] L. Debrauwer, A. Riu, M. Jouahri, E. Rathahao, Jouanin I., Antignac J.P., Cariou R., Le Bizec B., Zalko D., J. Chromatogr. (2005) 1082, 98-109. [3] L. Debrauwer, C. Marteau, D. Zalko, S. Chevolleau, Spectra Analyse (2010) 276, 38-44. [4] A. Riu, D. Zalko, L. Debrauwer, Rapid Commun. Mass Spectrom. (2006) 20, 2133-2142.
137/381
[P51] Quantification of total ApoE and ApoE4 isoform by targeted mass spectrometry across a clinical cohort (n=800): a case study for SRM-based assay validation and methionine-containing proteotypic peptide.
Romain Simon1, Catherine Fonbonne1, Jean Charles Lambert 2, Philippe Amouyel2, Jean Philippe Charrier3, Arnaud Salvador1, Jrme Lemoine1
1 2 3
Institut des Sciences Analytiques UMR 5280, Bd 11 Novembre 1918, 69622 Villeurbanne Institut Pasteur de Lille, Lille Centre, 59800 Lille bioMrieux, Chemin de l'Orme, 69280 Marcy l'Etoile
Contact: Jrme Lemoine (jerome.lemoine@univ-lyon1.fr)
Keywords: Clinical proteomics, Proteins, peptides and small molecules quantification Cerebrospinal fluid and plasma apoE levels have been proposed as diagnostic biomarkers to discriminate patients with Alzheimer disease (AD) against normal ederly. However, controversy remains about the clinical significance of plasma apoE levels due to inconsistency across the correlations between those patients, which might arise from different ELISA protocols and/or size and demographic cohorts. Coupling between liquid chromatography and targeted mass spectrometry, in the so-called selected reaction-monitoring mode (LC-SRM), has been shown to be an effective and orthogonal alternative to immuno-enzymatic tests for absolute protein quantification in biofluids. In the present study, we have developed SRM-based assays of total ApoE and ApoE4 isoform in plasma to address the lack of consensus regarding the possible association between ApoE and ApoE4 genotype and AD. AQUA standardization was carried out with the heavy forms of proteotypic sequence LGPLVEQGR tracking total ApoE and of allele specific sequence LGADMEDVR monitoring ApoE4 isoform, respectively. Robustness and precision of the workflow have been extensively evaluated over a one-month period, with 12 technical replicates per weak carried out by two experimentalists. Statistics on the replicates show CVs better than 10% and a lower limit of quantification of 1 microgram/mL for the less sensitive peptide sequence LGADMEDVCGR specific of ApoE4 allele. The validated SRM-based assay was then applied to the absolute quantification of total ApoE and ApoE4 isoform across a clinical cohort of 800 patients.
138/381
[P52] IDENTIFICATION DE SUBSTRATS DENZYMES DE MODIFICATION DES ARN PAR SPECTROMTRIE DE MASSE MALDI-TOF et MALDI-TOF/TOF
Vincent Gurineau1, Amandine Guelorget2, Djemel Hamdane2, Batrice Golinelli2, David Touboul1, Alain Brunelle1
1
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
2
Centre de Recherche de Gif, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France Contact: Vincent Gurineau (vincent.guerineau@icsn.cnrs-gif.fr)
Keywords: Organic and inorganic mass spectrometry, Systems biology Pour accder la base quelles modifient, les enzymes de modification des ARN retournent le nuclotide cible et plusieurs nuclotides voisins. Bien que de nombreuses structures cristallographiques de ces enzymes en complexe avec un ARNt aient rvl ce retournement de la base, la cintique rapide du retournement du nuclotide cible par des enzymes de modification des ARN na jamais t tudie par stopped-flow de fluorescence. En revanche, plusieurs tudes cintiques de ce phnomne ont t ralises sur des enzymes de modification de l'ADN. Cette approche est base sur lutilisation dun analogue fluorescent de ladnine, la 2aminopurine, comme indicateur des changements conformationnels de la structure de l'ADN. Pour tudier le mcanisme de retournement de la base par les m1A58 mthyltransfrases dARNt de T. thermophilus et de P. abyssi (TthTrmI et PabTrmI), la premire tape tait de trouver un mini- ARN substrat des enzymes TrmI. Les adnines 57 ou 58 de ce mini-substrat sont remplaces par une 2-aminopurine (2AP57 et 2AP58) et les deux mini-ARN ainsi obtenus sont tests comme substrats de TthTrmI et PabTrmI. Le mini-ARN 29-mer est incub avec l'enzyme TthTrmI ou PabTrmI. La raction est stoppe par ajout dun mlange phnol/chloroforme/alcool isoamylique pour prcipiter la protine. L'ARNt est extrait par centrifugation et prcipitation thanolique puis dessal sur une colonne MicroSpin G-25. L'ARNt est ensuite digr par la RNase A. Les oligonuclotides rsultants sont analyss par spectromtrie de masse MALDI-TOF en mode rflectron positif. La matrice DHB est choisie et le mode de dpt dried droplet utilis. Les oligonuclotides modifis dintrt (incrment de masse d aux mthylations supposes) sont tudis par spectromtrie de masse MALDI-TOF/TOF en mode MS et MS/MS (gaz de collision Ar, P=4,6x10-6 hPa, nergie de collision 2 keV). Le spectre de masse du produit de digestion du mini-ARN partir de l'chantillon tmoin (non incub avec l'enzyme) contient tous les pics attendus confirmant la squence nuclotidique. Aprs incubation avec l'enzyme TthTrmI, le spectre MS montre un ion m/z 1326,17 correspondant loligonuclotide AAAUp monomthyl. Le spectre MS/MS indique que la mthylation est localise uniquement sur ladnine 58, comme lindiquent les ions fragments m/z 997,18 (y3) et 673,14 (c2). Aprs incubation avec PabTrmI le fragment AAAUp est dimthyl et le spectre MS/MS montre que ces mthylations sont localises sur les adnines 57 et 58. Nos rsultats montrent que lARN 29-mer est un substrat de TthTrmI et de PabTrmI. Nous avons galement dtermin que la 2-aminopurine nest pas mthyle par les enzymes TrmI. Ceci nous permettra par la suite de diffrencier la mthylation des autres tapes catalytiques de faon pouvoir attribuer les vitesses correspondant la liaison de lARN lenzyme, puis du retournement de la base par stopped-flow de fluorescence.
139/381
[P53] Etude de la distribution spcifique de sels de benzalkonium par imagerie par spectromtrie de masse : Application la pharmacocintique oculaire chez le lapin.
Grgory Hamm1, Nicolas Desbenoit2, Raphael Legouffe1, Christophe Baudouin2, Alain Brunelle3, Isabelle Fournier4, Olivier Laprvote3, Maxence Wisztorski4, Franoise Brignole-Baudouin2, Jonathan Stauber1
1 2 3
ImaBiotech, Campus Cit Scientifique, 59655 Villeneuve dAscq, France Institut de la Vision, INSERM, UMR_S968, 17 rue Moreau, F-75012 Paris, France
Institut de Chimie des Substances Naturelles, Centre de recherche de Gif, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
4
Laboratoire de Spectromtrie de Masse Biologique Fondamentale et Applique (FABMS), EA 4550, Universit de Lille, 59655 Villeneuve dAscq, France
5
Chimie Toxicologie Analytique et Cellulaire, EA 4463, Facult des Sciences Pharmaceutiques et Biologiques, Universit Paris Descartes, 4 Avenue de lObservatoire, 75006 Paris, France Contact: Grgory Hamm (hamm.gregory@imabiotech.com)
Keywords: Imaging mass spectrometry

Les chlorures de benzalkonium (BAK) sont les conservateurs les plus largement utiliss dans les collyres, prsents gnralement sous forme de sel de benzododecinium (C12) et myristalkonium (C14). Ils permettent damliorer la pntration des composs actifs. Cependant, certaines tudes ont report un possible effet toxique de ces derniers au niveau de la surface oculaire et plus particulirement dans le cadre de traitements de longue dure comme celui contre le glaucome [1]. Ainsi, limagerie par spectromtrie de masse est utilise afin de caractriser la distribution spatiale des BAKs et valuer leur impact physiologique au niveau molculaire. Cette tude a t ralise sur des yeux de lapin instills avec des solutions de BAKs pendant diffrents temps de cintique (1 ou 5 mois) et nous as permis de dtecter deux types dions m/z 304.32 et m/z 332.36 correspondant respectivement au BAK C12 et C14. Une approche multi-technique a t choisie afin de mener ces travaux utilisant la spectromtrie de masse MALDI-TOF, le TOF-SIMS [2] ou encore le MALDI-Orbitrap. Cela a permis de faciliter la dtection dun large panel de composs (petites molcules, lipides [2], peptides, etc) et daccder aux plus hautes rsolutions spectrale et latrale. Cette prsentation sera consacre plus prcisment aux rsultats dimagerie MALDI-TOFMS. Nous discuterons de la localisation spcifique et de laccumulation des BAKs dans certaines zones histologiques dintrt (corne, angle iridocornen, conjonctive, limbe,) connues pour tre des rgions inflammatoires de lil. Lapport de la haute rsolution latrale sera dmontr sur deux rgions de lil particulirement importantes que sont le trabculum et le nerf optique. Nous pointerons galement une diffrence fine de comportement des deux types de BAK suivant leur structure. De plus, leur prsence sera valide par des tudes structurales par spectromtrie de masse en tandem ou assimiles. Par exemple, le mode FAST-SRM permettra de suivre un fragment spcifique dun compos, dans notre cas, m/z 212.42 (fragment du BAK C12) et ainsi valider sa distribution au niveau des structures oculaires prcdemment cites. Des expriences croises ont galement t menes entre deux laboratoires utilisant diffrents spectromtres de masse MALDI-TOF et modes de dposition de matrice afin de valider ces donnes. Limagerie par spectromtrie de masse apparait donc comme un outil de choix pour ltude de la distribution de composs connus pour avoir des effets dltres et peut tre utile dans le cadre dtude prclinique dans les domaines pharmacologique et toxicologique. 1 Baudouin, C., Detrimental effect of preservatives in eyedrops: implications for the treatment of glaucoma. 2008, Blackwell Publishing Ltd. p. 716-726. 2 Desbenoit, N., SMAP 2011, Avignon
140/381
[P54] Polymres synthtiques groupement terminal fragile : pr-traitement de lchantillon avant analyse MALDI
Christophe Chendo1, Caroline Barrre2, Valrie Monnier1, Thomas Trimaille3, Trang N.T. Phan3, Didier Gigmes3, Stphane Viel2, Stphane Humbel4, Laurence Charles2
1
Fdration des Sicences Chimiques - CNRS, FR1739, Spectropole, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille
2
Laboratoire Chimie Provence, UMR 6264, Equipe Spectromtries Appliques la Chimie Structurale, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille
3
Laboratoire Chimie Provence, UMR 6264, Equipe Chimie Radicalaire, Organique et Polymres de Spcialit, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille
4
Institut des Sciences Molculaires de Marseille, UMR 6263, Equipe Chimie Thorique et Mcanismes, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille Contact: Christophe Chendo (christophe.chendo@univ-cezanne.fr)
Keywords: Organic and inorganic mass spectrometry Les techniques de polymrisation radicalaire contrle (PRC) sont trs largement utilises pour synthtiser divers types de polymres de faibles indices de polydispersit en matrisant leur taille. Parmi ces techniques, la polymrisation assiste par nitroxyde (NMP) repose sur le pigeage rversible de radicaux polymres par lespce radicalaire stable quest le nitroxyde. Parmi les radicaux les plus utiliss, le nitroxyde SG1 (N-(2-mthylpropyl)-N-(1dithylphosphono-2,2-dimthyl propyl)-N-oxyl) permet la synthse de copolymres blocs amphiphiles de type poly(oxyde dthylne)/polystyrne mais aussi la croissance de segments acrylates. Cependant, lhomolyse rversible de la liaison C-ON qui est la base du processus NMP devient un inconvnient majeur lorsquil sagit de caractriser ces macromolcules par spectromtrie de masse aprs ionisation MALDI. En effet, le nitroxyde SG1 devient un groupement terminal labile, limin sous forme radicalaire lors du processus MALDI et lanalyse des groupements terminaux nest plus fiable. Une prcdente tude a montr que lorsque le processus dionisation favorisait la protonation spcifique de lazote du nitroxyde, la liaison C-ON liant SG1 au polymre tait renforce et des adduits cationiques intacts produits en phase gazeuse. Toutefois, les polymres synthtiques prsentant une affinit protonique dautant plus faible que leur taille augmente, cette mthode reste limite ltude de petits oligomres. Cette tude montre quune modification chimique du groupement terminal lissue de la polymrisation permet de renforcer la liaison C-ON et de conserver le groupement terminal lors de lanalyse MALDI. Le traitement du polymre par lacide trifluoroactique permet la substitution, par un atome dhydrogne, du groupement tert-butyl port par lazote du nitroxyde. La production dadduits intacts du polymre ainsi modifi montre que laugmentation de lnergie de dissociation de la liaison C-ON est effective et suffisante pour rsister laugmentation dnergie interne associe au processus MALDI. Cette mthodologie a t appliqu avec succs lanalyse des polymres de type polystyrne, poly(oxyde dthylne) et poly(butylacrylate).
141/381
[P55] Complmentarit des mthodes GC-MS/EI et LC-MS/APPI pour la comparaison de 29 rsines vgtales
Boutayna Rhourri-Frih1, Laure Fanton2, Patrice Andr2, Caroline West1, Michel Lafosse1, Patrick Chaimbault3
1
Institut de Chimie Organique et Analytique (ICOA) UMR CNRS 6005 Universit d'Orlans, Rue de Chartres - BP 6759, 45067 Orlans Cedex 2, France
2 3
LVMH Recherche, 185, Avenue de Verdun, 45804 Saint Jean de Braye, France
Laboratoire de Spectromtrie de Masse et de Chimie Laser (LSMCL). UPV-M .ICPM, 1, Boulevard Arago, 57078 Metz Technopole Cedex 03, France Contact: Patrick Chaimbault (patrick.chaimbault@univ-metz.fr) Keywords: Separative analysis methods, Organic and inorganic mass spectrometry Les rsines sont des mlanges complexes contenant un grand nombre de composs terpnodes de structures proches dont certains ont des proprits biologiques intressantes. Lanalyse de ces rsines a t dj largement ralise par GC-MS/EI (1-3) mais les donnes (temps de rtention et fragments m/z) sont abondantes et laborieuses utiliser pour comparer rapidement un grand nombre de rsines. Par ailleurs il est parfois difficile de caractriser une rsine nouvelle par manque de disponibilit des standards. Etant donn la provenance varie des 29 rsines prsentes dans ce travail (Inde, Cote divoire, Madagascar, Pakistan, Afghanistan), la diversit des familles, genres et espces (Gardenia gummifera, Daniellia oliveri, Boswellia serrata et dalzielli, Commiphera mukul et Canarium madagascariensis) dont elles sont issues et leur complexit (diterpnes, triterpnes, sesquiterpnes) voluant selon lge de la rsine, il a t ncessaire, pour les comparer, dutiliser deux mthodes chromatographiques couples la spectromtrie de masse de faon orthogonale (GC-MS/EI et LC-MS/APPI). Pour exploiter le grand nombre dinformations gnres par ces mthodes, nous avons utilis les diagrammes 3D bulles pour une comparaison rapide des rsines. La mthode danalyse par GC-MS/EI a t ralise aprs silylation de lextrait. Le diagramme 3D bulles reprsente en 2D chaque compos selon son temps de rtention et les valeurs m/z de chacun de leur fragment, chaque signal m/z tant reprsent par une bulle dont la grosseur dtermine lintensit. Ainsi dans un groupe de rsines il est possible de comparer celles qui sont similaires, celles qui ont des composs en commun et celles qui sont trs diffrentes de faon qualitative et quantitative. Pour complter ltude, le mme raisonnement est appliqu aux rsultats obtenus par LC-MS/APPI dont la dtection a t prcdemment optimise pour analyser les composs triterpnoides (4). Ces rsultats confirment ou infirment ceux de GC-MS/EI. 1- C. Math, G. Culioli, P. Archier, C. Vieillescazes, Characterization of archaeological frankincense by gas chromatographymass spectrometry, J. Chromatogr. A, (2004), 1023, 277-285 2- Laura Osete-Cortina, Mara Teresa Domenech-Carb Analytical characterization of diterpenoid resins present in pictorial varnishes using pyrolysisgas chromatographymass spectrometry with on line trimethylsilylation Journal of Chromatography A, (2005), 1065 265278 3- A. Otto, B. Simoneit, V. Wilde, L. Kunzmann, Wilhelm Pttmann Terpenoid composition of three fossil resins from Cretaceous and Tertiary conifers Review of Palaeobotany and Palynology (2002), 120, 203-215 4- B. Rhourrhi-Frih, P. Chaimbault, B. Claude, C. Lamy, P. Andr, M. Lafosse, Analysis of pentacyclic triterpenes by LC-MS. A comparative study between APCI and APPI, J. Mass Spectrom. (2009), 44, 71-80
142/381
[P56] Comparison of different liquid chromatography systems for the detection of low-level biomarkers in biological fluids by MRM
Florence Roux-Dalvai1, Violette Gautier1, Alexandre Stella1, Jrme Lemoine2, Anne Gonzalez de Peredo1, Bernard Monsarrat1, Odile Burlet-Schiltz1
1
Institut de Pharmacologie et de Biologie Structurale, CNRS, UMR5089, Universit de Toulouse, 205, route de Narbonne , 31077 Toulouse, France
2
Institut des Sciences Analytiques, UMR 5280 CNRS, Universit de Lyon, , 69622 Villeurbanne, France
Contact: Florence Roux-Dalvai (florence.dalvai@ipbs.fr)
Keywords: Clinical proteomics, Separative analysis methods, Proteins, peptides and small molecules quantification Detection of low-abundant proteins in biological samples is usually hampered by limited sensitivity and dynamic range of mass spectrometers. In the Selected Reaction Monitoring (SRM) mode, the dynamic range problem is alleviated, as target parent and fragment ions are respectively selected in a narrow mass range window by the two quadrupole analyzers, filtering out other highly abundant peptide ions that otherwise would lead to signal suppression in a full MS scan. Nonetheless, SRM analysis of weakly concentrated molecule results in low ion currents in the mass spectrometer, and intrinsic sensitivity of the instrument may then become the limiting factor of the analysis. Injecting higher amounts of analyte in the mass spectrometer could thus in this case help to improve the detection of species present at very low level in a complex matrix. Here, we tested different sample processing methods and LC-MS instrumental settings to optimize the detection of biomarker candidates in two biological fluids. Increasing amounts of sample were loaded either on a 75m I.D. capillary column coupled to a nanospray source, or on a 2mm I.D. column coupled to an ESI source. In each case, several proteins were monitored by SRM on a 5500QTrapTM mass spectrometer (ABSciex), in order to determine the optimal loading capacity of the chromatographic system. In the case of the nanoLC setting, samples were loaded on C18 precolumns with different loading capacity, and gradients of different lengths were tested in order to increase the amount of material loaded on the system without affecting the linearity of the MS response. For each experimental setting, the limit of detection by SRM of candidate biomarkers, spiked at different concentrations in plasma or cerebrospinal fluid, was determined. Our results indicate that the loss of sensitivity of conventional HPLC compared to nanoLC is balanced by the higher amount of sample that can be injected in the instrument, making this instrumental configuration an interesting alternative for the detection of low-abundant species, as long as the absolute quantity of starting material does not represent a limiting factor.
143/381
[P57] Dtection rapide de toxines de la menace NRBC sur surfaces par DESI-MS
Alexandre Seyer1, Franois Becher1, Franois Fenaille1, Sandra Alves2, Jean-Claude Tabet2
1 2
CEA, iBiTec-S, Service de Pharmacologie et dImmunoanalyse, , 91191 Gif-sur-Yvette, France
Universit Pierre et Marie Curie - Institut Parisien de Chimie Molculaire, Chimie Structurale Organique et Biologique (CSOB), (UMR 7201), 4, place Jussieu, 75252 Paris cedex 05, France Contact: Alexandre Seyer (alexandre.seyer@cea.fr)
Keywords: High mass and high resolution mass Spectrometry Parmi tous les agents lis au bioterrorisme rpertoris par le Center of Disease Control (CDC, Atlanta, USA), les toxines protiques comme la ricine (issue de Ricinus communis) ou la toxine epsilon (issue de Clostridium perfringens) sont considres comme des armes potentielles. Nanmoins, les mthodes de dtection de ces toxines ncessitent souvent de longues tapes de prparation dchantillon, allongeant ainsi le temps de rponse. Celles bases sur la spectromtrie de masse font notamment appel des approches de reconnaissance anticorps/antignes et de digestion enzymatique trs sensibles mais pouvant demander plusieurs heures ( 16h).[1] Notre tude consiste rduire au maximum ces tapes de prparation dchantillon et dtecter ces protines directement sur plusieurs types de surfaces, afin de rendre lanalyse ralisable un plus haut dbit. Ces objectifs pourraient tre atteints grce lutilisation dun spectromtre de masse haute rsolution et prcision de mesure de masse (LTQ-Orbitrap, Thermo Scientific, San Jose, CA) coupl une source dionisation/dsorption pression atmosphrique de type DESI (Desorption Electrospray Ionization; Prosalia, Indianapolis, IN). La capacit de la source DESI dtecter des petites molcules (explosifs, drogues, pesticides) a dj t largement explore,[2] mais son utilisation pour la dtection de toxines de hautes masses molculaires na pas encore t mise en valeur. Ainsi, nous avons valu deux approches diffrentes. Une premire, mettant laccent sur la rapidit et la simplicit danalyse, a consist dtecter les toxines de manire intacte, et une seconde, plus sensible, faisant appel une tape de digestion enzymatique in situ. Les paramtres exprimentaux, i.e. la composition du faisceau de gouttelettes primaires, la gomtrie de la source DESI et le capillaire de transfert (capillaire ESI ou nez DESI), influenant le transfert des gouttelettes/agrgats secondaires dans le spectromtre de masse, ont t optimiss dans chaque cas (protines intactes ou mlange complexe de peptides). Les rsultats montrent quune dizaine de femtomoles dune protine modle de 17 kDa dpose sur une surface hydrophobe, peuvent tre dtectes sans aucun traitement de lchantillon. Il a aussi t possible didentifier quelques picomoles dune protine de 66 kDa aprs seulement deux heures de digestion enzymatique in situ. Lobjectif est maintenant dappliquer ces mthodes des toxines protiques prsentes dans diverses matrices complexes (eau de boisson, lait) et sur diffrents types de surfaces (cartons, plastiques). [1] Duriez, E.; Fenaille, F.; Tabet, J.C.; Lamourette, P.; Hilaire, D.; Becher, F.; Ezan, E. J. Proteom. Res. 2008, 7, 4154-63. [2] Takts, Z.; Wieman, J.M.; Cooks, R.G. J. Mass Spectrom. 2006, 40, 1261-75.
144/381
[P58] Etude du Profil de Mthylation de lHistone 3 (H3.1) par Spectromtrie de Masse

Magalie Duchateau1, Monica Rolando2, Carmen Buchrieser2, Abdelkader Namane1
1 2
Plate-Forme de Protomique, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France
Unit postulante de Bioloie des Bactries intracellulaires, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France Contact: Magalie Duchateau (magalie.duchateau@pasteur.fr)
Keywords: Clinical proteomics, Signaling, interactomics and post-translational modifications Cette tude sintresse une protine recombinante de Legionella pneumophila qui a la capacit de modifier les histones des cellules de lhte. Les histones sont de petites protines basiques sujettes de nombreuses modifications post-traductionnelles (MPT) comme les actylations, les phosphorylations ou encore les mthylations. La mthylation affecte particulirement les rsidus lysine et arginine, induisant des diffrences de masse de 14 Da (mono-mthylation), 28 Da (di-mthylation) ou 42 Da (tri-mthylation). Les variations dexpression de ces MPTs sont connues comme jouant un rle clef dans de nombreux processus biologiques (activation , rpression de la transcription). Lapproche bottom-up classiquement utilise en protomique consiste digrer la protine dintrt par une enzyme, comme la trypsine, et analyser les peptides rsultant de cette digestion enzymatique par spectromtrie de masse couple la chromatographie liquide (LCMS/MS). Bien que suffisante pour la majorit des protines, la digestion trypsique prsente des limitations pour ltude des histones. En effet, les histones sont des protines extrmement riches en rsidus lysine et arginine conduisant lobtention de nombreuses petites squences peptidiques monocharges dont les spectres MS/MS sont difficilement interprtables. Ces rsidus sont, de plus, particulirement localiss du ct N-terminal des histones, l o le phnomne de mthylation est principalement attendu. Afin de dterminer prcisment le profil de mthylation de lhistone H3.1, deux stratgies complmentaires ont t mises en uvre : (i) lutilisation denzymes protolytiques, autre que la trypsine, comme la lysine C vitant ainsi le clivage au niveau des rsidus arginine, (ii) lutilisation dun peptide de synthse correspondant aux 21 premiers acides amins de notre protine dintrt. Ce peptide, une fois la raction de mthylation ralise, a t tudi par approche top-down et les efficacits de plusieurs modes de fragmentation ont t compares (CID, HCD et ETD). Cette combinaison dapproches nous a permis didentifier et de localiser les rsidus ayant subi une ou plusieurs mthylations. Link: http://genopole.pasteur.fr/PF3
145/381
[P59] Differential proteomic 2d-dige analysis of failing left ventricles in mice overexpressing fkbp12.6
Patricia ROUET-BENZINEB1, Christian FEDERICI3, Miresta PREVILON1, Morgane LE GALL2, Guilhem CLARY2, Cdric BROUSSARD3, Franois GUILLONNEAU3, Philippe CHAFEY2, Jean-Jacques MERCADIER1
1 2 3
Inserm U698, Universit Paris Diderot, 46 rue Henri Huchard, 75018 Paris Plateforme protomique Institut Cochin, 22 rue Mchain, 75014 Paris Plateforme protomique Universit Paris Descartes, 22 rue Mchain, 75014 Paris
Contact: Philippe CHAFEY (philippe.chafey@inserm.fr)
Keywords: Systems biology In cardiac myocytes, FKBP12.6 (FK506 binding protein or calstabin 2) binds to and modulates the activity of the ryanodine receptor type 2 (RyR2), the Ca2+-channel of the sarcoplasmic reticulum (SR). FKBP12.6 is thought to favour synchronous RyR2 opening during systole and to prevent SR calcium leak during diastole. Increased Ca2+ leak through RyR2 during diastole has been implicated in the pathophysiology of heart failure (HF). We made the hypothesis that, by altering SR Ca2+ homeostasis, FKBP12.6 overexpression would influence Ca2+-dependent signalling pathways during pressure overload-induced HF. In addition, RyR2 may not be the only cellular target for FKBP12.6 in cardiomyocytes. We used our mouse model of cardiac specific conditional overexpression of FKBP12.6. Wild type (WT) mice and mice overexpressing FKBP12.6 (DT) were submitted to transverse aortic constriction (TAC) and compared at 30 days to sham-operated mice of the same genotype. We analyzed total proteins extracted from the LV of 48 mice of both gender (8 WT sham-operated (WT sham), 16 WT TAC, 8 FKBP12.6 shamoperated (DT sham), and 16 FKBP12.6 TAC (DT TAC)). TAC mice were selected for the presence of congestive heart failure according to the presence of pulmonary oedema (defined as the ratio of lung weight to tibia length > lung weight to tibia length in sham-operated mice + 3 SD). Quantitative differential proteomic analysis was performed using 2D-DIGE (2 DimensionalDifferential In-Gel Electrophoresis). Data processing and spot quantification were performed using DeCyder software (GE Healthcare Bio-Sciences). Spots differentially expressed in either group as compared to the others (WT Sham vs WT TAC, DT Sham vs DT TAC, WT Sham vs DT Sham, WT TAC vs DT TAC) were identified by tandem mass spectrometry. Our results show that there are a large number of differentially expressed proteins between WT and DT failing left ventricles. Whatever the alteration (up or down regulation), the effect appeared less pronounced in DT TAC than in WT TAC vs the respective sham group. Our Results will be described and discussed.
146/381
[P60] Analyses de xnobiotiques dans une matrice aqueuse pour valider un systme dlimination de ces composs
Marion Martignac1, Samuel Pollet2, Mat Maurette1, Esther Oliveros1, Jacques Debuire2, Florence BenoitMarqui1, Catherine Claparols3
1
Laboratoire des IMRCP (UMR CNRS 5623), Universit Paul Sabatier (Toulouse III), 118 route de Narbonne, 31062 Toulouse Cedex 9
2 3 4
Lora, Bt II, 1 rond point de Flotis, 31240 Saint Jean Laboratoire de Chime de Coordination, 205 route de Narbonne, 31077 Toulouse Cedex 4
Serv. Commun de Spectromtrie de Masse, universit Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9 Contact: Catherine Claparols (claparol@chimie.ups-tlse.fr)
Keywords: Organic and inorganic mass spectrometry, Separative analysis methods Les xnobiotiques, substances chimiques trangres aux organismes vivants qui proviennent des utilisations humaines et vtrinaires [1], sont prsents dans les eaux de diffrentes origines. Les rejets des industries chimiques et pharmaceutiques ainsi que ceux des tablissements de soins, les eaux uses collectes par les gouts mais aussi les rejets dlevages industriels, contiennent des xnobiotiques dorigine mdicamenteuse [2]. Gnralement, non totalement limines dans les stations dpuration [3], ces substances sont rejetes dans le milieu naturel quelles perturbent avant de se retrouver nouveau dans le circuit dalimentation et de distribution deau potable [4,5]. Les impacts que les xnobiotiques peuvent avoir sur la sant humaine sont encore mal connus mais leurs consquences mme trs faible concentration (de lordre du ng/L) sur le comportement et la biologie de la faune piscicole sont dj avres [6]. Pour lutter contre ce phnomne, la socit Lora exprimente un nouveau procd (appel Lolyse), qui combine un bioracteur membranes avec un racteur photochimique. Dans une phase dvaluation prliminaire, certaines molcules organiques ont t choisies parmi celles rfrences dans la littrature [7] (paractamol, ibuprofne, diclofnac, 5FU, carbamazpine, parabne). Il sagissait de vrifier la dgradation de solutions synthtiques de ces molcules par le procd Lolyse. Les dgradations ont t effectues sur des solutions de concentrations variables de composs purs et en mlanges; les solutions synthtiques ont t prpares dans de leau du robinet, de leffluent de station dpuration et de leffluent hospitalier. Les dgradations sont suivies en LC/MS/MS (Q-Trap Applied Biosystems). La LC/MS/MS est la technique de prdilection en raison de sa polyvalence, de sa spcificit et sa slectivit. Les tests que nous avons effectus montrent que la dgradation est pratiquement totale (concentrations non quantifiables, infrieures au seuil de dtection (ng/L)) en seulement quelques minutes. De plus, la carbamazpine qui nest pas du tout limine par les traitements des stations dpuration [5,8] est dgrade par cette mthode.
1. Virvoulet, G., Environnement Risques et Sant 5, 239-241 (2006) 2. Langford K.H. et Thomas K.V., Environment International 35, 766-770 (2009) 3. Piram A., Salvador A., Gauvrit J-Y, Lanteri P. et Faure R., Talanta 74, 1463-1475 (2008) 4. Mompelat S., Le Bot B. et Thomas O., Environment International 35, 803-814 (2009) 5. Togola A. et Budzinski, H., Journal of Chromatography A 1177, 150-158 (2008) 6. Lange A., Parell G.C., Coe T.S., Katsu T.S., Urushitani Y., Iguchi T., Tyler C.R., Environmental Science & Technology 43, 1219-1225 (2009) 7. Besse J.P., Garric J., Toxicology Letters 176, 104-128 (2008) 8. Heberer T., Journal of Hydrology 266, 175-189 (2002)
147/381
[P61] Minimization of back-exchange in a top-down ecd/etd h/d exchange approach for protein structural characterization
Ophlie Robine1, Edith Nicol1, Guillaume van der Rest1, Julia Chamot-Rooke1
1
Laboratoire des Mcanismes Ractionnels, CNRS, Ecole Polytechnique, route de Saclay, 91128 Palaiseau, France Contact: Ophlie Robine (ophelie@dcmr.polytechnique.fr)
Keywords: High mass and high resolution mass Spectrometry The association of hydrogen/deuterium exchange and mass spectrometry (HDX MS) emerged in past years as a powerful experimental tool capable of probing both structural and dynamic features of proteins. However, this approach suffers some important limitations due to the occurrence of (i) back-exchange, which can take place at different stages of the experiment, and (ii) scrambling between protons and deuterons in the gas phase, especially in MS/MS experiments. These processes have to be minimized in order to get interpretable results. In the course of the development of a top-down ECD HDX method based on the ECD fragmentation in the gas phase of the entire deuterated protein, difficulties arising in experimental reproducibility led us to analyze more deeply the different factors influencing the extent of back exchange as observed in the spectra. Indeed, although it has been show that ECD or ETD [1, 2] can minimize scrambling (intramolecular hydrogen migration process before backbone cleavage), back exchange that can occur before the fragmentation step can lead to uninterpretable results. However, back exchange is not easy to track since it can occur at different stages of the experimental procedure, from the quench step to the last desolvation process to form ions in the gas phase. In this work, we studied the different parameters having a potential influence on the backexchange with the aim to find a set of optimized parameters allowing for robust HDX MS/MS experiments. The parameters tested were: solvent composition, temperature, flow rate and type of ion source. Several modes of sample introduction were studied : ice cooled syringe, which was first described by Pr. Jrgensen, as well as a home-made cryosource which is an air-locked box kept at low temperature (-30C) where samples are stored before their introduction in the mass spectrometer. This study led us to a set of optimized experimental conditions that were further used for the analysis of peptides specifically designed for HDX experiments and small proteins. Our results clearly indicate that the minimization of back-exchange is a key parameter for successful HDX MS/MS experiments, providing robust and confident conditions for the analysis. [1] K. Rand, et. al., Electron transfer dissociation facilitates the measurement of deuterium incorporation into selectively labeled peptides with single residue resolution, J. Am. Chem. Soc., (2008), 130(51), 17453-17459 [2] J. Pan, et. al., Electron capture dissociation of electrosprayed protein ions for spatially resolved hydrogen exchange measurements, J. Am. Chem. Soc.,(2008), 130(35), 11574-11576
148/381
[P62] Fragmentation patterns of unesterified bisphosphonates by electrospray tandem mass spectrometry

Erwann Gunin2, Thierry Jouenne1, Julie Hardouin1
1 2
Laboratoire PBS -UMR CNRS 6270, Universit de Rouen, 76821 Mont-Saint-Aignan cedex Laboratoire CSPBAT , 74 rue Marcel Cachin, 93017 Bobigny cedex
Contact: Julie Hardouin (julie.hardouin@univ-rouen.fr)
Keywords: Systems biology Bisphosphonates are important drugs for the treatment of a variety of bone diseases. Since some of these compounds have no chromophore, their detection is challenging and mass spectrometry (MS) appears to be an appropriate sensitive tool. Previous studies were done by ESI-MS(n) in positive and negative modes for studying bisphosphonates. The compounds were analyzed by ESI-MS(n) (1,2). Following the side chain and the esterification functions, different fragmentation pathways were deduced. Here, new unesterified bisphosphonates were synthesised. Our work deals with the analysis by ESI-MS(n) of these unesterified bisphosphonates on both an ion trap (to carry out MS(n) experiments (until n=5)) and a Q-TOF mass spectrometer (to have better mass accuracy) in positive and negative modes. The dissociation mechanisms of these prodrugs were elucidated both in positive and in negative ion modes. We presented here complete fragmentation fingerprints of the unesterified bisphosphonates both in positive and negative modes by ESI-MS(n). We showed the influence of the side chain on the fragmentation. Moreover the use of D2O allowed us to check the fragmentation pathways from the mass spectra acquired on a LTQ Velos Orbitrap. These fingerprints will be of great value for differentiating these potential prodrugs in complex biological mixtures. (1) Hardouin, Gunin, Monteil, Caron, Lecouvey, Journal of Mass Spectrometry 2008, 43: 10371044. (2) Hardouin, Gunin, Malosse, Caron, Lecouvey, Rapid Communications in Mass Spectrometry 2008, 22: 2287-2300.
149/381
[P63] Protomique fonctionnelle de la signalisation du rcepteur TrkA dans la rponse au NGF versus proNGF dans le cancer du sein
Cyril Corbet1, Lo Aubert1, Hubert Hondermarck1, Xuefen Le Bourhis1, Robert-Alain Toillon1
1
INSERM U908 Signalisation des fecteurs de croissance dans le cancer du sein-Protomique fonctionnelle, Cit Scientifique Btiment SN3, 3me tage, 59655 Villeneuve d'Ascq, France Contact: Cyril Corbet (cyril.corbet@ed.univ-lille1.fr)
Keywords: Signaling, interactomics and post-translational modifications Notre laboratoire a dmontr que le Nerve Growth Factor (NGF) est scrt par les cellules cancreuses de sein et quil induit leur prolifration, leur survie et leur migration/invasion suivant une boucle autocrine. Nous avons rcemment mis en vidence que son prcurseur, le proNGF, est aussi produit et scrt par les cellules de cancer du sein. Longtemps dcrit comme inactif biologiquement, les effets du proNGF dans les rponses cellulaires ont t sous-estims voire ignors. Quelques travaux commencent les dcrypter. Ainsi, dans les cellules neuronales, le proNGF est dcrit comme un inducteur dapoptose. Dans ce contexte, lobjectif de nos travaux consiste dissocier et caractriser les effets du proNGF par rapport au NGF sur les cellules cancreuses de sein. Grce lutilisation de siRNA et dinhibiteurs pharmacologiques spcifiques, nous avons montr que, bien quagissant via le rcepteur tyrosine kinase TrkA, le NGF et le proNGF induisent des cascades de transduction diffrentes. Nous avons donc recherch les partenaires de ce rcepteur par protomique fonctionnelle. Lanalyse protomique LC-MS/MS a permis didentifier des protines diffrentiellement recrutes en fonction du ligand, avec lexistence nouvelle de co-rcepteurs membranaires. Ces interactions sont en cours de validation par des techniques dimmunoprcipitation inverse et de colocalisation en microscopie confocale et leur rle dans lactivation des voies de transduction est apprhend par inhibition par siRNA et/ou anticorps bloquants. Ainsi, lensemble de nos rsultats obtenus par cette approche de protomique fonctionnelle montrent que les voies du NGF et du proNGF, bien quapparemment identiques, sont au contraire significativement dissociables. La discrimination de ces deux voies offre ainsi la possibilit de nouvelles modulations thrapeutiques dans le cancer du sein.
150/381
[P64] Identification systmatique du peptidome exocellulaire de Lactococcus lactis

Mylne Boulay1, Alain Guillot2, Vronique Monnet1, Vincent Juillard1
1
Equipe Peptides et Communication Bactrienne, INRA, UMR1319 MICALIS, Domaine de Vilvert, 78352 Jouy en Josas
2
Plate-Forme d'Analyse Protomique de Paris Sud-Ouest, INRA, UMR1319 MICALIS , Domaine de Vilvert, 78352 Jouy en Josas Contact: Alain Guillot (alain.guillot@jouy.inra.fr)
Keywords: Separative analysis methods La dtection des peptides dans un milieu de culture bactrien reprsente un enjeu important. Certains sont potentiellement impliqus dans divers phnomnes physiologiques tels que linduction de la virulence ou la communication cellulaire. Leur caractrisation s'est gnralement faite en suivant des dmarches cibles s'appuyant sur leur purification. Notre objectif est de caractriser de faon systmatique l'ensemble des peptides produits par la bactrie lactique Lactococcus lactis IL1403 pendant sa croissance dans un milieu chimiquement dfini ne contenant pas de peptides au dpart. La base de donnes gnomiques utilise a t enrichie avec les petites squences codantes, gnralement mal dtectes, et qui ont t spcifiquement recherches in silico dans le gnome de L. lactis. Les peptides sont concentrs et pr-purifis sur la base de leur hydrophobicit et leur taille avant dtre spars par 2D-LC off line sur phase inverse de type C18. La premire sparation chromatographique est ralise pH neutre. Chacune des fractions collectes est ensuite spare pH acide en couplage un spectromtre de masse LTQ-Orbitrap. Lidentification des peptides est ralise par empreinte de fragmentation contre la base enrichie via X!tandem et Bioworks. Plus de 1500 squences peptidiques distinctes ont t identifies. Elles drivent en majorit de protines (> 10 kDa) de surface et, dans une moindre mesure, de protines cytoplasmiques, ce qui reflte une activit protolytique de surface. Des peptides fortement hydrophobes correspondant des fragments de squences signal dadressage sont galement identifis. Pour des raisons encore indtermines, peu de squences attribues aux petits gnes sont dtectes.
151/381
[P65] Synthesis and structural characterisation of pamam and modified pamam using mass spectrometry
Emma-Dune LERICHE1, Gael Coadou1, Corinne Loutelier-Bourhis1, Martin Grossel 2, Catherine Lange1
1
Universit de Rouen, UMR 6014, COBRA, FR CNRS 3038, Rue tesnire, 76821 Mont-saint-Aignan cedex, France
2
University of Southampton, School of chemistry, Highfield, Hants SO17 1BJ Southampton, UK
Contact: Emma-Dune LERICHE (lericheemmadune@yahoo.fr)
Keywords: Ionic mobility, Separative analysis methods Polyamidoamine (PAMAM) dendrimer have grown in popularity in the past decade in variety of disciplines. This can be attributed to their application in biomedicine and their capacity to be used as nonviral gene delivery vector1. Indeed, PAMAM can function as highly efficient cationic vectors for delivering genetic material into cells. However, the shortcoming of these compounds is their quite significant cytotoxicity linked to their cationic surface. To decrease toxic effects and increase efficiency as drug and gene carrier of dendrimers, functionalization with hydrophobic groups on surface can be performed to minimize the overall charge density. The modification of PAMAM surface has been described in literature which related papers focus on biological aspect and cytotoxicity2,3. For this study, full generations of PAMAM (G0, G1) with ammonia and ethylenediamine (EDA) core4 activity and minimize cytotoxicity are under progress5. Synthesis and chemical modifications are performed according to 4-5. The structural characterization and identification of the synthesized dendrimers are performed by Electrospray/Multi-stage Mass Spectrometry (ESI/MSn)6. The purity and monodispersity of individual whole generation were monitored using capillary electrophoresis (CE), hydrophilic interaction liquid chromatography (HILIC) and Ion-mobility spectrometry (IMS). 1- Roseita Esfand and al., Drug discovery today, 2001, 6, 427-436. 2- R. Jevprasesphant and al., International Journal of Pharmaceutics, 2003, 252, 263-266 3- R. B. Kolhatkar and al., Bioconjugate Chem. 2007, 18, 2054-2060 4- 5- D. A. Tomalia and al., Polymer Journal, 1985, 17, 117-132 5- X. Wang and al., Biomacromolecules, 2010, 11, 245251 6- T. J.-C. Vincent, R. Dol and C. M. Lange, Rapid commun. Mass Spectrom. 2008, 22: 363-372
152/381
[P66] Investigation of in-source decay of oxime-linked peptide by MALDI-TOF-MS

Julie Hardouin1, Galle Anne Cremer 1, Agns Delmas1
1 2
Centre de Biophysique Molculaire - UPR 4301 CNRS, rue Charles Sadron, 45071 Orlans Cedex, France Laboratoire PBS - UMR CNRS 6270, Universit de Rouen, 76821 Mont-Saint-Aignan cedex, France
Contact: Julie Hardouin (julie.hardouin@univ-rouen.fr)
Keywords: Organic and inorganic mass spectrometry Chemoselective ligation methods which allow for the condensation of unprotected peptide fragments under mild aqueous conditions without any activation step have been of growing importance in the last decade for the preparation of peptide conjugates. These bioconjugation approaches are based on reactions belonging to the click chemistry repertoire. Oxime ligation is one of the most preferred procedures, due to the high chemoselectivity between aminoxyconjugates and an aldehyde or a ketone provided that the high reactivity of aminooxy-linked peptides towards carbonyl-containing compounds and the N-overacylation of the NH-O group are well controlled. To characterize and improve these new procedures, it was necessary to reliably identify all the compounds associated with the targets. MALDI-TOF-MS is the preferred method for the routine characterisation of peptide-based conjugates because of its high sensitivity, rapidity, and simplicity of operation. Although MALDI is a soft ionization technique, a significant degree of decay behavior can occur in the source. In-source decay (ISD) is directly observed after desorption/ionization step in the source region (1-3). However, some prompt fragmentations of labile proline-containing peptides observed in a MALDI source cannot be understood as a classical ISD fragmentation as recently highlighted (4). UV laser irradiation can also lead to the photodissociation of photolabile molecules during MALDI analysis and product ions are detected in their mass spectra (5). Oxime-containing peptides are known to absorb UV irradiation in solution but, despite the use of oxime bioconjugation, only few articles have noticed that the oxime bond may dissociate during a MALDI analysis. We have therefore decided to investigate, in gas phase, the fragmentation of peptides containing an oxime bond by MALDI-TOF-MS. The goal of this study is to propose structures of these product ions generated in the MALDI source. For this purpose, different compounds were synthesized: two linear peptides with only one oxime bond and two branched peptides containing two oxime bonds. (1)Brown and Lennon, Anal Chem, 1995, 67, 3990-3999. (2)Hardouin, Mass Spectrom Rev, 2007, 26, 672 682. (3)Takayama, J Am Soc Mass Spectrom, 2001, 12, 420-427. (4)Sachon, Clodic, Blasco, Jacquot, Bolbach, Anal Chem, 2009, 81, 8986-8992. (5)Low, Kang, DiGruccio, Kirby, Perrin, Fischer, J Am Soc Mass Spectrom, 2004, 15, 1156-1160.
153/381
[P67] Application of Electrospray Charge-Detection Mass Spectrometry to the Characterization of Megadalton Sized Macromolecules and Block Copolymer Micelles
Tristan Doussineau1, Cong Yu Bao1, Wenjing Zhang2, Franck DAgosto2, Bernadette Charleux2, Rodolphe Antoine1, Philippe Dugourd1
1
LASIM-UMR 5579 CNRS / UCBL, Domaine Scientifique de la Doua, Universit Claude Bernard Lyon 1, Btiment Alfred Kastler, 43 Bd du 11novembre 1918, 69622 Villeurbanne, Cedex
2
LCPP/C2P2-UMR 5265 CNRS/UCBL, Bt. CPE, 43 Bd du 11novembre 1918, 69622 Villeurbanne, Cedex
Contact: Rodolphe Antoine (rantoine@lasim.univ-lyon1.fr)
Keywords: Instrumentation Mass is a critical parameter for characterization and identification of biological molecules, complexes, assemblies, and particles. The development of electrospray ionization (ESI) sources, has led to widespread applications of mass spectrometry in modern biomedical and biotechnology research. Very large objects, such as proteic complex assemblies and viruses were analyzed by electrospray mass spectrometry. However, the extension of ESI to the megato gigadalton size regime is trickier due to the large amount of charge that can be carried by such ions. Charge detection mass spectrometry (CD-MS) measures simultaneously the charge and the velocity of individual multicharged macroions allowing the determination of their respective mass. An innovative mass spectrometer based on charge detection has recently been developed in Lyon. In particular, the device was used to study poly(ethyleneoxide) (PEO) samples with average molecular weight ranging from 1,000,000 to 7,000,000 Da. This single event technique allowed mass and charge distributions to be determine for each sample as well as their respective polydispersity index. Moreover, 3D charge-vs-mass image were plotted allowing the investigation of the charging of these electrosprayed macroions as a function of concentration and nature of alkali cations present in sample solutions. Self-assembled nanometric micelles prepared via RAFT-mediated emulsion polymerization of styrene using a poly(methacrylic acid-co-PEG methacrylate) macroRAFT agent with a trithiocarbonate reactive group have also been studied with the developed instrument. Mass distributions and polydispersity index have been compared to those obtained with conventional techniques.
154/381
[P68] Faire son choix parmi la multitude des techniques d'analyse d'un phosphoprotome : application celui de Bacillus subtilis.
Thophile Cocquerez1, Lauriane Kuhn1, Philippe Hammann1
1
Plate-forme Protomique Strasbourg Esplanade, Institut de Biologie Molculaire et Cellulaire, FRC1589, 15 rue Ren Descartes, 67084 Strasbourg Cedex Contact: Lauriane Kuhn (proteomic-esplanade@unistra.fr)
Keywords: Signaling, interactomics and post-translational modifications, Separative analysis methods Le dveloppement des plate-formes technologiques pour l'analyse des protines par spectromtrie de masse permet la communaut scientifique de disposer d'quipements de qualit pour raliser des prestations de service et des collaborations dans le domaine de la protomique. Notre plate-forme, situe sur le site de l'Esplanade Strasbourg, est sollicite la fois pour identifier les protines contenues dans un chantillon plus ou moins complexe, mais aussi pour caractriser plus finement les ventuelles modifications portes par des protines d'intrt. Les phosphorylations sont l'une des modifications qui rencontrent le plus d'intrt. En effet, la phosphorylation des protines est l'un des mcanismes de rgulation le plus frquent et le plus important, ayant un rle prpondrant dans un trs grand nombre de processus cellulaires. Cette modification post-traductionnelle, rencontre sur les rsidus srine, thronine et tyrosine, a trs tt sduit la communaut scientifique utilisant la spectromtrie de masse, donnant ainsi lieu au dveloppement des stratgies d'analyse cibles pour sa dtection et sa caractrisation. Pour rpondre une demande croissante d'analyse de protines phosphoryles, nous avons donc orient nos dveloppements sur ce sujet. Notre but est de pouvoir disposer de techniques d'analyse pour mettre en vidence la prsence de phosphorylations sur des protines isoles ou sur un extrait protique plus complexe, l'aide de techniques d'enrichissement de type MOAC et de la spectromtrie de masse disponible sur le site (MALDI-TOF/TOF et nanoLC-MSMS). Le matriel biologique utilis pour raliser ces essais a t compos la fois de standards protiques (Beta-caseine, mlange de peptides synthtiques phosphoryls ou non) et d'un extrait protique comprenant les protines solubles de Bacillus subtilis. L'tude prsente ici a consist comparer les stratgies suivantes : (i) analyse off-gel en utilisant une approche nanoLC-MS/MS, (ii) une sparation par SDS-PAGE couple des analyses nanoLC-MS/MS et finalement (iii) une sparation par lectrophorse 2D couple des analyses par MALDITOF(/TOF). Pour chacune de ses 3 stratgies, les chantillons ont t : (i) soit analyss directement sans traitement pralable, (ii) soit aprs l'utilisation d'une technique d'enrichissement commerciale de type MOAC (comparaison de 3 kits commerciaux sous forme de cnes ou de spin-colonnes), (iii) soit avec l'utilisation de colorants rvlant spcifiquement la prsence de protines phosphoryles sur gel d'lectrophorse.
155/381
[P69] Differential and quantitative analysis of dog oviductal fluid

Grgoire Harichaux1, Valrie Labas1, Marie Saint-Dizier3, Sylvie Chastant-Maillard3, Karine Reynaud3
1
INRA, Plate-forme d'Analyse Intgrative des Biomarqeurs, Centre de Recherches INRA de Tours, 37380 Nouzilly
2
INRA, UMR INRA 85, Physiologie de la Reproduction et des Comportements UMR CNRS 6175 Universit de Tours -IFCE, Centre de Recherches INRA de Tours, 37380 Nouzilly
3
ENVA, UMR 1198 INRA-ENVA Biologie du Dveloppement et Reproduction, 7 Avenue du Gnral de Gaulle, 94700 Maisons-Alfort Contact: Grgoire Harichaux (gregoire.harichaux@tours.inra.fr)
Keywords: Proteins, peptides and small molecules quantification The major reproductive peculiarity of the bitch is that ovulation releases prophase I (Germinal Vesicle, immature) oocytes. Resumption of meiotic maturation, as well as fertilisation and embryonic development to the morula stage occur in the oviduct. The dog is a biomedical model of human diseases and also a model for endangered canid species, so the development of reproductive biotechnologies may be of great interest. In many mammalian species, embryos and offspring can be obtained in vitro. However, in the canine, in vitro-produced embryos remain exceptional and no puppy has been born to date after IVF. The main limiting factor of in vitro embryo production remains the in vitro oocyte maturation process and mean MII rates are usually around 1020%. A better knowledge of the composition of oviductal fluid may help to mimic the in vivo conditions during in vitro maturation, fertilisation and embryonic development. The objective of this study was to analyse the oviductal fluid by a label-free quantitative proteomic workflow based on SDS-PAGE protein separation, nanoLC-MS/MS analysis and quantitative method using spectral counting. Oviductal fluids were collected from 3 bitches, 3.5 days after ovulation (moment of oocyte maturation in the oviduct). Two oviductal regions (ampulla and isthmus) were dissected and flushed with 50l of PBS. Oviductal fluids were submitted to 1D SDS-PAGE and all bands were digested with trypsin. Peptide extracts were analysed on an Ettan MDLC system (GE HealthCare) coupled to a linear ion trap LTQ mass spectrometer (Thermofisher). After protein identification using Mascot server with SwissProt and NCBI databases, bioinformatic processing of data and statistic analysis (T-test with p value < 0.05) were performed using the spectral counting quantitative module of the Scaffold software. Using this strategy, more than 420 proteins were qualitatively identified in canine oviductal fluid and the gene ontology analysis displayed biological pathways specific to the biology of reproduction. Quantitatively, few proteins were differentially expressed between ampulla and isthmus. Now, this list of protein candidates may to be validated by immonodetection methods.
156/381
[P70] FragMixer : un Programme pour lIdentification de Phosphopeptides partir de deux Modes de Fragmentation MS/MS
Vronique Hourdel1, Mathias Vandenbogaert 2, Olivia Jardin-Math2, Jean Bigeard3, Heribert Hirt3, Benno Schwikowski2, Delphine Pflieger4
1 2 3 4
Institut Pasteur, Plate-forme de Protomique, 28 rue du Docteur Roux, 75015 Paris Institut Pasteur, Laboratoire de Biologie Systmique, CNRS URA 2171, 28 rue du Docteur Roux, 75015 Paris URGV Plant Genomics, INRA/CNRS/Universit dEvry Val dEssonne, rue Gaston Crmieux, 91057 Evry
Laboratoire Analyse et Modlisation pour la Biologie et lEnvironnement, UMR CNRS 8587, Universit dEvry Val dEssonne , bd Mitterrand, 91025 Evry Contact: Delphine Pflieger (delphine.pflieger@univ-evry.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics, Signaling, interactomics and post-translational modifications Identifier par spectromtrie de masse des peptides phosphoryls est encore une entreprise difficile, parce que les spectres de fragmentation obtenus sur ces espces ne contiennent pas toujours suffisamment dions pour tablir avec certitude la squence en acides amins et les sites prcis de phosphorylation. La combinaison de deux spectres de fragmentation a t dcrite comme une option pertinente afin damliorer lidentification des phosphopeptides, par exemple en utilisant diffrents modes de fragmentation CID, ETD et/ou HCD disponibles sur les appareils hybrides comme le LTQ-Orbitrap. Les squences identifies partir des deux spectres obtenus sur le mme prcurseur doivent alors tre fusionnes en une unique squence consensus, en termes de squence en acides amins et de site(s) de phosphorylation. Dans la prsente tude, nous avons valu, sur des chantillons de phosphopeptides analyss sur un appareil LTQ-Orbitrap, lintrt dacqurir sur chaque prcurseur slectionn des spectres MS2 et MSA (MultiStage Activation) dans la trappe linaire, en dpit de la forte similitude de ces deux spectres. Nous avons dvelopp FragMixer, un programme qui dtermine la squence consensus partir des identifications fournies par Mascot sur les paires de spectres. Le Mascot Delta score (1), diffrence de scores entre la premire squence thorique attribue un spectre MSn et la suivante, a t rcemment valid sur des phosphopeptides synthtiques comme permettant de discriminer les sites de phosphorylation corrects partir des rsultats didentification de Mascot. FragMixer, qui offre plusieurs options pour filtrer les squences de peptides exportes, utilise essentiellement le Mascot Delta score afin dtablir les squences phosphoryles consensus partir des paires de scans acquises, en conservant automatiquement lincertitude de position de phosphate si ncessaire. FragMixer permet de traiter les rsultats didentification Mascot obtenus sur les paires de spectres MS2/MSA testes dans cette tude, mais aussi dautres paires de spectres qui requirent des paramtres diffrents de recherche dans les banques (par exemple MSA/ETD ou ETD/HCD). (1) Savitski MM et al. Confident phosphorylation site localization using the Mascot Delta Score. Mol Cell Proteomics. 2011 Feb;10(2):M110.003830.
157/381
[P71] Validation de la levure S. cerevisiae comme modle dtude de la translecture.

David Cornu1, Sandra Blanchet2, Manuela Argentini3, Jean-Pierre Rousset 2, Olivier Namy2
1 2 3
FRC 3115, ICSN, avenue de la Terrasse, 91190 Gif-sur-Yvette Institut de Gntique et Microbiologie, Universit Paris-Sud 11, 15 Rue Georges Clemenceau, 91405 Orsay IFR 115, ICSN, avenue de la Terrasse, 91190 Gif-sur-Yvette
Contact: David Cornu (david.cornu@icsn.cnrs-gif.fr)
Keywords: Proteins, peptides and small molecules quantification La terminaison de la traduction correspond la dernire tape de la synthse protique. Elle se produit lorsquun codon stop entre dans le site A du ribosome et fait intervenir deux facteurs protiques eRF1 et eRF3. La terminaison est un processus fidle. Toutefois, il existe des squences sur certains ARNm permettant de reprogrammer le ribosome afin de provoquer une erreur traductionnelle. Parmi les erreurs traductionnelles possibles, la translecture de codon stop correspond lincorporation dun ARNt charg au niveau du codon stop, permettant la poursuite de la traduction dans la mme phase jusqu'au codon stop suivant. D'aprs les rgles du code gntique nous pouvons prdire l'existence de plusieurs ARNt suppresseurs naturels capables de dcoder un codon stop. L'efficacit d'incorporation de chacun de ces ARNt dpend non seulement de la complmentarit codon stop/anticodon mais aussi du contexte nuclotide entourant le codon stop. Afin de mieux comprendre les mcanismes molculaires de la translecture, nous avons dtermin par spectromtrie de masse la nature des acides amins incorpors lors de la translecture chez Saccharomyces cerevisiae. Nous avons mis au point un nouveau systme rapporteur permettant de purifier spcifiquement les protines issues de la translecture chez la levure. Ce systme rapporteur est bas sur l'utilisation de la GST dont la squence codante est interrompue par un codon stop. Ainsi seule les protines obtenues par translecture pourront tre purifies. Les protines sont purifies par chromatographie daffinit, proteolyses et les mlanges peptidiques sont analyss par spectromtrie de masse MALDI, nanoLC-MS/MS et nanoLC-MS. Les recherches des peptides chevauchant le site de translecture sont effectues en utilisant une banque constitue des 20 squences GST qui diffrent entre elles par la nature de lacide amin positionn au niveau du codon stop dans la squence de translecture. Pour un contexte nuclotidique donn, les acides amins majoritairement insrs sont la glutamine et la tyrosine pour les codons stops UAA et UAG et la cystine et le tryptophane pour le codon UGA. Des analyses quantitatives ont permis de montrer que l'efficacit relative dincorporation des acides amins Q et Y varie selon les codons stop. Ces rsultats confirment que plusieurs ARNt naturels ont la capacit de dcoder les codons stop chez la levure. De plus nos rsultats dmontrent que la nature du msappariement entre le codon stop et l'anticodon de l'ARNt joue un rle majeur dans l'efficacit d'incorporation de l'ARNt suppresseur naturel. Ces rsultats valident la pertinence de la levure S. cerevisiae comme modle pour ltude de la translecture. Link: https://www.imagif.cnrs.fr/plateforme-23-Service_d %E2%80%99identification_et_de_caract%C3%A9risation_des_prot%C3%A9ines_par_spectrom %C3%A9trie_de_mass
158/381
[P72] Imagerie par spectrometrie de masse tof-sims et etude immuno-histologiques appliquees a lophtalmologie : etude des effets du chlorure de benzalkonium
Nicolas Desbenoit1, Christophe Baudouin1, Olivier Laprvote4, Alain Brunelle2, Franoise Brignole-Baudouin1
1 2
Institut de la Vision, INSERM, U968, 17 rue Moreau, 75012 Paris
Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la terrasse, 91198 Gif-sur-Yvette
3 4
Centre Hospitalier National dOphtalmologie des Quinze-Vingts Paris, 28 rue de Charenton, 75571 Paris
Chimie Toxicologie Analytique et Cellulaire, EA 4463, Facult des Sciences Pharmaceutiques et Biologiques, Universit Paris Descartes, 4 Avenue de lObservatoire, 75006 Paris Contact: Nicolas Desbenoit (nicolas@icsn.cnrs-gif.fr)
Keywords: Imaging mass spectrometry Grce leurs proprits dsinfectantes, les chlorures de benzalkonium (BAK) sont des conservateurs essentiellement employs dans les collyres o ils sont rputs pour favoriser la pntration des principes actifs. Les effets cytotoxiques sur la surface oculaire de faibles doses en utilisation chronique sont dsormais bien connus et concernent de manire cruciale les patients glaucomateux qui sont obligatoirement traits vie pour viter la ccit. Ainsi, la distribution des BAKs dans lil mrite dtre rvalue laide de limagerie par spectromtrie de masse (IMS), afin de comprendre les impacts de cette toxicit sur des structures primordiales dans la pathologie glaucomateuse. LIMS est une technique de choix pour lanalyse en surface des tissus biologiques. En effet cette technique permet en une simple acquisition de localiser de nombreux composs biologiques. Les deux techniques dimagerie par spectromtrie de masse les plus couramment utilises aujourdhui sont le ToF-SIMS et le MALDI. Les rsultats obtenus par IMS dans le cadre du consortium Masda-Eye, et qui font lobjet dune autre prsentation, montrent que le BAK est retrouv dans de nombreuses zones histologiques de lil, en particulier le nerf optique [1]. Ce rsum a pour objet de mettre en valeur lapport de limagerie ToF-SIMS pour caractriser les variations lipidiques gnres par le BAK, avec une haute rsolution spatiale de lordre du micromtre, et afin de montrer les consquences physiopathologiques au niveau molculaire en termes de lipidomique. Des yeux de lapins albinos ont t instills avec une solution de BAK 0,01% pour une dure de cinq mois. Aprs sacrifice, les yeux ont t rapidement nucls, et conservs -80C dans une gomme tragacanthe. Des coupes sries de 14 m dpaisseur ont t ralises -30C laide dun cryostat. Les images ioniques sont obtenues avec un spectromtre de masse ToF-SIMS. Les images de 500x500 m (rsolution spatiale de 2 m) ont t enregistres en mode dionisation positif. Nous avons montr une forte corrlation spatiale entre le BAK (deux ions dtects correspondant respectivement au benzododecinium m/z 304,32, et au myristalkonium m/z 332,36) et un antioxydant, la vitamine E, dans le cul-de-sac conjonctival. Les images ioniques ont indiqu une distribution la priphrie extrieure du globe oculaire. La corrlation spatiale entre les images ioniques et limmunohistologie a mis en vidence les zones de mort cellulaire. En conclusion, limagerie par ToF-SIMS ouvre dimportantes perspectives pour lvaluation fine de la toxicit lie la prsence de la molcule en termes de lipidomique et de mtabolomique. [1] G. Hamm et al., SMAP 2011, Avignon.
159/381
[P73] Identification par une approche protomique des protines membranaires de Plasmodium falciparum exprimes la surface de l'rythrocyte, en fonction de son affinit pour un rcepteur d'adhsion
Gwladys Bertin1, Franois Guillonneau2, Virginie Salnot2, Nadine Fievet3, Philippe Deloron1
1 2 3
UMR216 mre et enfant face aux infections tropicales, 4 avenue de l'observatoire, 75006 Paris, France Plate-forme protomique Paris Descartes, 22 rue mchain, 75014 Paris, France UMR216 mre et enfant face aux infections tropicales, 08 B.P 841, Cotonou, Bnin
Contact: Gwladys Bertin (gwladys.bertin@gmail.com)
Keywords: Systems biology Le paludisme est prsent en Afrique Sub-Saharienne et est particulirement dangereux chez les jeunes enfants et les femmes enceintes. Il est admis que la virulence du parasite est lie sa capacit exprimer des antignes variable de surface (VSA). Ces derniers prsentent une affinit pour diffrents rcepteurs de l'hte, impliqus dans la physiopathologie des formes graves (paludisme gestationnel et crbral). Lors de sa maturation, le parasite met en place un trafic protique consquent au niveau membranaire et sub-membranaire de l'rythrocyte. Cet adressage protique donne lieu la formation de knobs permettant l'expression des VSA dont PfEMP1, protine au rle majeur dans la gravit de l'infection. Ces PfEMP1 sont des protines minoritaires noyes dans une masse protique rythrocytaire prsentant un haut de degr d'insolubilit. Toutes les tudes du protome membranaire de Plasmodium n'ont permis d'identifier les VSA, et notamment PfEMP1, qu'avec un nombre de peptides restreint et de faibles scores. Notre tude, actuellement en cours, repose sur un comparatif 2 2 d'isolats parasitaires d'accs simple, crbral ou gestationnel visant la structure des knobs et les VSA, dont PfEMP1. Lobtention dextraits protiques issus disolats de terrain en fait loriginalit et permettra didentifier la ou les PfEMP1 impliques dans les accs pernicieux. Actuellement, nous avons optimis les protocoles de prparation d'chantillons savoir un enrichissement de 90% des formes matures du parasite par colonne macs et une dpltion des hmaties saines. Le culot cellulaire est lys par choc osmotique et les protines sont solubilises avec 2% SDS et digres en solution par la trypsine. Les peptides sont analyss par nano LC-Orbitrap. Nous avons identifi partir disolats placentaires et daccs simple 530 protines parasitaires dont 1/3 issues de la membrane parasitaire et/ou rythrocytaire, dont PfEMP1 avec un score de 70 avec 2 peptides. Ces rsultats sont encourageants pour la suite de l'tude au vu de la littrature.
160/381
[P74] Etude allergomique du pollen de cyprs

Youcef SHAHALI1, Jean-Pierre SUTRA1, Iman HADDAD1, Jolle VINH1, Philippe CHAFEY2, Sylvie CHOLLETMARTIN3, Denis CHARPIN4, Adriano MARI5, Hlne SENECHAL1, Pascal PONCET1
1 2 3 4 5
ESPCI, LSABM et LSBMP, 10 rue Vauquelin, 75005 Paris, France INSERM ICGM, 22 rue Mchain, 75014 Paris, France INSERM, Hpital Bichat, 46 rue Henri Huchard, 75018 Paris, France Hpital Nord de Marseille, Chemin des Bourrely, 13915 Marseille Cedex 20 CCEA, via dei Monti di Creta, 104 I-00167 Rome, Italie
Contact: Youcef SHAHALI (youcef.shahali@espci.fr)
Keywords: Clinical proteomics, Systems biology, Separative analysis methods INTRODUCTION: L'analyse du rpertoire des allergnes et des familles molculaires d'allergnes permet de distinguer les polysensibilisations des ractions croises et est la base des progrs du diagnostic de l'allergie. L'allergie au pollen de cyprs (Cupressus sempervirens), principale pollinose en rgion mditerranenne la symptomatologie parfois trs svre, touche prs de 10% des enfants et sa prvalence est en augmentation. OBJECTIF: Etablir le rpertoire des allergnes de C. sempervirens et celui de la rponse IgE des patients allergiques. Mettre en vidence des corrlations entre les donnes cliniques et des profils lectrophortiques d'immunoractivit IgE. METHODE: L'analyse allergomique drive de la protomique. Les protines, extraites en conditions aqueuses ou en dtergent partir de pollen de cyprs, sont spares par lectrophorse 2D ou 2 x 1D, technique rcemment applique lallergomique. Elles sont ensuite transfres sur nitrocellulose et incube avec des srums de patient. Les IgE fixes sur les allergnes sont ensuite rvles. Les spots protiques correspondants aux allergnes sont prlevs et identifis par spectromtrie de masse. RESULTATS: Deux profils d'immunoractivit IgE sont observs (n=80). Une catgorie de patients reconnat des allergnes glycosyls de hautes masses molaires (HMW) et une deuxime catgorie, des motifs peptidiques d'une protine basique de 14 kDa. L'allergne majeur Cup s 1 (43kDa, pI 7,0) est retrouv parmi les allergnes HMW et nous dcrivons, pour la premire fois, la polygalacturonase de C. sempervirens 43kDa de pI basique (Cup s 2). La protine de 14 kDa semble spcifique du pollen de C. sempervirens. Elle pourrait tre de type lectinique et prsente des homologies avec le facteur dlongation de lhva, Hev b 1, allergne majeur du latex. Les six peptides internes squencs en LC/MS/MS couvrent 52% de la squence de Hev b 1. De plus, une ractivit IgE autoanticorps dirige contre des neutrophiles humains a pu tre mise en vidence seulement dans les srums de patients reconnaissant la protine de 14 kDa. CONCLUSION: Ltude de ce modle dallergie dbouche sur des problmatiques dordre analytique (nouveaux allergnes) et mcanistique (ractions croises/inflammation/autoimmunit). Le squenage complet de la protine de 14kDa de C. sempervirens ainsi que sa pertinence clinique dans l'inflammation, dans l'autoimmunit ainsi que dans la dgranulation des basophiles restent prciser.
161/381
[P75] Towards complete sets of plant proteins by positional proteomics approaches: expanding and integrating the knowledge of N- and C- protein modifications
Thierry Meinnel1, Willy Bienvenut2, Wojciech Majeran1, Carmela Giglione1
1 2
CNRS, ISV, UPR2355, Btiment 23A, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette CNRS, ISV, FRC3115, Btiment 23A, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette
Contact: Thierry Meinnel (thierry.meinnel@isv.cnrs-gif.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications Novel proteomic approaches combining (i) low abundant proteomes biochemical enrichment, (ii) in vivo labeling with reactive precursors to specifically tag some posttranslational modifications, and (iii) powerful separation or peptide purification methods, now make it possible to select either N- or C-terminal peptides [1-5]. Analysis of the selected peptides by state-of-the-art mass spectrometry with the concept of one peptide-one protein provided MS/MS data are convincing enough - leads to the identification of weakly abundant and very small proteins which otherwise are undetectable in classic proteomics. Quantification is possible between various samples and within each sample when dealing with the various states of protein modifications. With such approaches applied to plant proteomes, it is now possible to deepen our knowledge to the less abundant proteins and to identify the many protein variants displaying N- and C-modifications. Such modifications frequently including acylation and proteolytic events are essential to guide the fate of any protein and route them to the proper cell compartment where they are supposed to exert their activity. The workflow of such an approach - as now developed in our laboratory using Arabidopsis thaliana and Chlamydomonas reinhardtii [6]- will be presented and discussed here. We will also discuss the strong addedvalue of including bio-informatic tools in the pipeline for the sake of adding to the robustness of the conclusions. Finally, N- or C-lipidation are known to involve many major membrane signal transduction proteins - including many kinases and phosphatases as well as a wide range of cell membrane functions which specifically respond to different physiological conditions. Similar to phosphoproteomics - but restricted to a smaller number of candidates and reactive sites any variation of the N- and C-acylation proteome should lead to uncover new mechanisms in signal perception at the molecular level. System biology analysis using such data could be initiated as a result. Reference 1. Meinnel & Giglione (2008) Proteomics 8, 626-49. 2. Mischerikow & Heck (2011) Proteomics 11, 571-89. 3. Mohammed & Heck (2011) Curr Opin Biotechnol 22, 9-16. 4. Schilling et al. (2010) Nat Methods 7, 508-11. 5. Kleifeld et al. (2010) Nat Biotechnol 28, 281-8. 6. Bienvenut et al. (2011) Proteomics 11, 1734-50. Keywords: complete proteomes, proteolysis, lipidation, acetylation, cell compartments
162/381
[P76] Comparative large-scale characterisation of plant vs. mammal proteins: Divergences and convergences
Willy V. Bienvenut2, Christelle Espagne1, Aude Martinez1, David Sumpton3, Sergio Lilla3, Wojciech Majeran1, Thierry Meinnel1, Carmela Giglione1
1 2 3
CNRS, ISV, UPR2355, Btiment 23A, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette CNRS, ISV, FRC3115, Btiment 23A, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, G61 6BD Glasgow, UK
Contact: Willy V. Bienvenut (willy.bienvenut@isv.cnrs-gif.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications N-terminal modifications play a major role for the fate of proteins in terms of activity, stability, or subcellular compartmentalization [1, 2]. Such modifications remain poorly described and badly characterised in proteomic studies. Recent advances in the field allow now specifically enrich and select N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein Nterminal modifications. Here, we aimed at studying extensively such modifications in the model plant Arabidopsis thaliana and to compare these results to those obtained from a human sample run and analysed in parallel. We applied large-scale analysis to compile robust conclusions on both datasets. Our data show strong convergence for some of the characterised modifications between the mammal and plant Kingdoms such as N-terminal Methionine Excision or N-Myristoylation. Although proteins N-terminus acetylation show some similarities for substrates specificity in general, a large proportions of plant organelle-targeted proteins feature post-translational N-alpha-acetylation of the mature protein after removal of the transit peptide. Also, downstream alternative starts of translation appear to be frequent in humans but exceptional in plants. References 1. Martinez et al. (2008) Proteomics 8, 2809-31. 2. Meinnel & Giglione (2008) Proteomics 8, 626-49. Keywords: proteolysis, lipidation, acetylation, cell compartments
163/381
[P77] Dtection dEscherichia coli dans un milieu complexe par amplification phagique et spectromtrie de masse en mode MRM
Armelle Martelet1, Guillaume L'Hostis1, Eric Ezan2, Christophe Junot2, Christine Rozand3, Alain Theretz3, Gaspard Gervasi3, Jean-Claude Tabet4, Bruno Muller1, Franois Becher2
1
Equipe mixte CEA/bioMrieux, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, CEA Saclay, 91191 Gif-sur-Yvette, France
2
CEA, iBiTec-S, Service de Pharmacologie et dImmunoanalyse, Laboratoire d'Etude du Mtabolisme des Mdicaments, CEA Saclay, 91191 Gif-sur-Yvette, France
3 4
bioMrieux S.A., Chemin de lorme, 69280 Marcy-lEtoile, France
Equipe de Spectromtrie de masse, Institut Parisien de Chimie Molculaire, UMR 7201, Universit Pierre et Marie Curie-Paris 6, 4 place Jussieu, 75252 Paris Cedex 05, France Contact: Armelle Martelet (armelle.martelet@cea.fr)
Keywords: Clinical proteomics, High mass and high resolution mass Spectrometry, Proteins, peptides and small molecules quantification La dtection rapide despces bactriennes pathognes est cruciale en sant publique, et dans le domaine de la scurit alimentaire. Escherichia coli est une bactrie frquemment trouve dans les contaminations alimentaires, et dont certaines souches peuvent engendrer de graves problmes de sant et parfois entraner la mort. lheure actuelle, lidentification bactrienne ncessite une culture de plusieurs heures afin dobtenir une quantit de bactries compatible avec la sensibilit des mthodes de dtection. Il y a donc un rel besoin de dvelopper de nouvelles mthodes de dtection bactrienne la fois rapide et sensible. Notre objectif est de raccourcir significativement le temps de culture ncessaire la dtection bactrienne laide dune amplification phagique. Un bactriophage (ou phage) est un virus n'infectant que des bactries. Leur utilisation prsente plusieurs intrts pour la dtection bactrienne comme la spcificit de reconnaissance pour leur bactrie hte, qui peut aller de la souche bactrienne plusieurs genres bactriens. Un autre intrt est li leur multiplication au sein de la bactrie. En effet, en fonction de la taille du phage, linfection dune bactrie par un phage aboutit la libration de 102 104 particules virales no-formes, dans la plupart des cas aprs lyse de la bactrie hte. Le nombre de protines constituant le phage est donc multipli par le mme facteur et le phnomne d'amplification est dautant plus intressant quil se droule en seulement quelques dizaines de minutes. Dautre part, les phages utilisent la machinerie cellulaire de la bactrie hte pour se multiplier et offrent ainsi lavantage de pouvoir diffrencier les cellules viables et non viables. Nous avons choisi de combiner lutilisation des phages la spectromtrie de masse en tudiant le modle du phage lytique T4 et son hte naturel Escherichia coli. Ce phage, qui comporte plus de 300 protines, est l'un des plus complexes. Une analyse protomique par chromatographie liquide couple la spectromtrie de masse haute rsolution (Orbitrap) nous a permis d'identifier deux marqueurs de l'infection bactrienne par le phage T4 : une protine de la capside du virion (9 kDa), et une protine interne la tte du phage (8 kDa). Par la suite, nous avons dmontr la faisabilit de dtection de peptides prototypiques de ces deux marqueurs dans diffrentes matrices grce une approche cible par LC-MS/MS en mode Multiple Reaction Monitoring [MRM]. Nos premiers essais ont permis de dtecter 105 cfu/mL dE.coli en milieu de culture et 106 cfu/mL dans une matrice alimentaire complexe (cassoulet). Ces premiers rsultats, en cours damlioration, montrent le potentiel du couplage de lamplification phagique et de la spectromtrie de masse dans le dveloppement de techniques de dtection bactrienne dans le domaine de la scurit alimentaire.
164/381
[P78] Use of procaine and procainamide as co-matrices for the analysis of oligosaccharides by MALDI-TOF mass spectrometry
Hlne Lavanant1, Corinne Loutelier-Bourhis1
1
Universit de Rouen, UMR CNRS 6014 COBRA, FR 3038, rue Tesnire, 76821 Mont St Aignan CEDEX, France
Contact: Hlne Lavanant (helene.lavanant@univ-rouen.fr)
Keywords: Organic and inorganic mass spectrometry Reductive amination of oligosaccharides is widely used as a method to enhance sensibility and facilitate sequence determination. The derivation protocol involves one or two steps and requires purification of the excess reagents by extraction and reversed-phase HPLC. As an alternative, we used procaine and procainamide as co-matrices with 2,5-dihydroxybenzoic acid (DHB). We added a 10g/L procaine hydrochloride or procainamide hydrochloride solution in acetonitrile with 10% acetic acid to a maltooligosaccharide solution one minute before preparing our MALDI targets with DHB with the dried droplet method. This protocol resulted in deposits composed of very fine homogeneous crystals, contrary to DHB which tends to form large inhomogeneous crystals around the rim of the MALDI spot. As a result, mass spectra could easily be acquired in an automated mode and presented a very high percentage of derivatised oligosaccharides. The derivatised oligosaccharides were the unreduced Schiff base with a mass difference of 218,142 u for procaine and 217,158 u for procainamide. The presence of procaine or procainamide on the target did not impede the ionization and rather improved the signal to noise ratio. Mass spectra reacquired several days after sample preparation showed the Schiff base derivatives were more stable than the underivatised oligosaccharide, as the percentage of derivatised oligosaccharides increased with time. Although tests of enhancement of sensibility with a maltoheptaose standard were not very favorable, one advantage was found in the structural information derived from MALDI TOF/TOF experiments with procaine and procainamide derivatives. Indeed, while underivatised maltooligosaccharides could only be detected as sodium adducts MNa+, procaine derivatives were mainly observed as protonated molecules MH+. With MH+ precursor ions, MALDI TOF/TOF mass spectra showed exclusively y series ions because the proton was fixed on the basic Schiff base. When MNa+ ions of derivatised maltooligosaccharides were selected as precursor ions, both y and b series fragment ions could be observed, although with low intensity as the major fragmentation involved loss of the Schiff base. Link: ircof.crihan.fr
165/381
[P79] Proteomic analysis of glutathionylation in the phosynthetic bacteria, Synechocystis sp PCC 6803
Solenne CHARDONNET1, Samer SAKR2, Pierre LE MARECHAL1, Corinne CASSIER-CHAUVAT2, Paulette DECOTTIGNIES1
1
Institut de Biochimie et Biophysique Molculaire et Cellulaire, UMR 8619, Universit Paris-sud, Bt 430, 91405 Orsay cedex, France
2
iBiTec-S,SBIGeM, LBI, URA2096, CEA-Saclay, 91191 Gif-sur-Yvette, France
Contact: Solenne CHARDONNET (solenne.chardonnet@u-psud.fr)
Keywords: Signaling, interactomics and post-translational modifications Redox regulation of proteins through various modifications of cysteine residues is now widely recognized as an important regulatory process. Among these modifications, protein glutathionylation is emerging as a crucial mechanism for signal transduction and regulation of protein function, in mammals but also in photosynthetic organisms. This reversible posttranslational modification consists in the formation of a mixed-disulfide bridge between an accessible free thiol on a protein and a molecule of glutathione. Glutathionylation can protect specific cysteines from irreversible oxidation but can also modulate protein activities and thereby play a role in many cellular processes. Several methods have been developed to identify proteins undergoing gluthathionylation but they have been mostly applied to mammals while little is known in photosynthetic organisms. To investigate this mechanism at a large scale in the phosynthetic bacteria Synechocystis sp. PCC 6803, we used a method based on labelling with biotinylated glutathione (BioGSSG). BioGSSG was synthetized from hydroxysulfosuccinimidebiotin and glutathione, and incubated with a total soluble protein extract from Synechocystis. Glutathionylated proteins were purified by avidin affinity chromatography, separated on SDSPAGE and identified using nLC-CHIP-IT-MS/MS with the X!TANDEM search engine. This strategy allowed us to obtain the most complete list of in vitro glutathionylated targets (about 200) proposed in a photosynthetic organism up to now. These proteins are involved in several cellular processes, especially photosynthesis, amino-acid metabolism and protein biogenesis and degradation. Moreover, the sites of glutathionylation were determined for 70 proteins after trypsin cleavage. For one of these targets, peroxiredoxin II, glutathionylation of the recombinant protein with oxidized glutathione was confirmed by MALDI-TOF mass spectrometry, and its effect on the activity was studied.
166/381
[P80] Comparative analysis of histone post-translational modification patterns obtained by mass spectrometry on intact proteins, Asp-N/Glu-C and tryptic peptides
Kvin Contrepois1, Eric Ezan2, Carl Mann1, Franois Fenaille2
1 2
CEA, iBiTec-S, SBIGeM, CEA Saclay, 91191 Gif Sur Yvette, France CEA, iBiTec-S, SPI, CEA Saclay, 91191 Gif Sur Yvette, France
Contact: Franois Fenaille (francois.fenaille@cea.fr)
Keywords: Signaling, interactomics and post-translational modifications Histones are subjected to extensive post-translational modifications including mainly acetylation and methylation of lysine residues. These post-translational modifications (PTMs) are known to play key roles in DNA-based biological processes. We have recently developed a fast and reproducible method involving ultra-high performance liquid chromatography (UHPLC) coupled to a high resolution/high mass accuracy LTQ-Orbitrap mass spectrometer to characterize core histone modifications/variants from WI-38 primary human fibroblasts [1]. However, under these conditions, modifications with the same nominal mass (e.g., acetylation and tri-methylation) can not be unambiguously distinguished and site-specific information is lacking. To overcome these limitations and to further validate the data obtained on intact proteins, the results were compared to those from 'conventional reference methods' (middle-down and bottom-up approaches). Kelleher and coworkers have previously reported that solution ratios of defined mixtures of unmodified and acetylated H4 peptides (2 kDa) vary dramatically from the observed MS ratios (due to differences in ionization efficiency), while solution ratios of recombinant H4 protein mutants (11 kDa) correspond closely with MS ratios [2]. In this context, the aim of this work was to evaluate whether similar observations could be made on Asp-N, Glu-C (middle-down), and propionylated tryptic peptides (bottom-up), by comparison with data obtained at the protein level (top-down). The reproducibility of each approach was first assessed by performing independent biological replicate experiments. The in-depth study of histones H4, H2A.Z and H2A.O modifications revealed that similar relative abundances were observed, whatever the method used. Moreover, we will present examples indicating that these approaches are highly complementary and that the use of an integrated approach is required for optimal characterization of histone PTMs and variants. To the best of our knowledge, this study is the first comparative evaluation of those three distinct platforms. References [1] Contrepois et al, J Proteome Res 2010, 9, 5501-10. [2] Pesavento et al, Anal Chem 2006, 78, 4271-80.
167/381
[P81] Analysis of headspace of flavour compounds using PTR-ToF Mass Spectrometry: optimisation of acquisition parameters
Xavira Pennanec1, Etienne Smon1, Jean-Luc Le Qur 1
1
Centre des Sciences du Got et de l'Alimentation, UMR6265 CNRS, UMR1324 INRA, Universit de Bourgogne, Agrosup Dijon, 17 rue Sully, 21000 Dijon Contact: Xavira Pennanec (Xaviera.Pennanec@dijon.inra.fr)
Keywords: Instrumentation Food aroma is complex, normally composed of a mixture of volatile compounds in a wide range of concentration. Besides, temporal aspect of flavour release is an important part contributing to sensory impression during food consumption. Thus, real time techniques such as atmospheric pressure chemical ionisation (APCI-) or proton transfer reaction (PTR-) mass spectrometry have been widely applied to the measurement of in vivo volatiles release for in-nose measurement of the expired air composition. Proton transfer reaction-mass spectrometry (PTR-MS) experiments are based on volatile compounds chemical ionisation mainly using H3O+ as precursor ion. H2O is firstly ionised in the ion source to generate H3O+. Reagent ions then react with neutral molecules into the drift tube, mainly producing [MH]+ protonated ions. Ionisation is only possible for molecules with a proton affinity (PA) superior to that of water (PAWater = 691 kJ/mol). By varying E/N values (E: electric field strength in the drift tube; N: buffer gas number density in the drift tube), detection of analytes can be improved. The optimal E/N value is linked to the physico-chemical nature of the analyte. The aim of this work was to optimize proton transfer parameters for flavour compounds detection with our PTR mass spectrometer. Analyses were conducted on the headspace of eleven different aroma compounds belonging to various chemical classes, chosen for their frequent use in food flavouring. Signal response was followed according to varying E/N values, paying attention to molecular ions intensities and presence of fragment ions. The results highlighted that the E/N parameter is closely linked to the chemical structure of the ionised molecules. Using proton transfer reaction mass spectrometry, it clearly appears that it is crucial to control acquisition parameters and especially the E/N value to optimise the detection of flavour compounds in complex matrices such as food.
168/381
[P82] Modification d'un spectromtre de masse TSQ700 pour le dpt non destructif de molcules sous Ultra Vide
Audrey Bodin1, Pierre Abeilhou1, David Martrou1, Sbastien Gauthier1
1
Centre d'Elaboration de Matriaux et d'Etudes Structurales, 29 rue Jeanne Marvig BP94347, 31055 Toulouse Cedex 4 Contact: Audrey Bodin (audrey.bodin@cemes.fr)
Keywords: Instrumentation La technique de dpt gnralement utilise pour dposer des molcules sous vide est lvaporation thermique. Cependant cette technique est trop nergtique pour le dpt de molcules fragiles. Ltude des proprits de molcules adsorbes ncessite donc la mise au point de procds de dpt moins destructifs. Lionisation par lectrospray (ESI) est une mthode douce dionisation dveloppe pour ltude des protines. Lappareil commercial utilis pour notre tude est un spectromtre de masse Finnigan triple quadruple TSQ700. Il doit tre coupl un quipement multi-chambres, sous ultra haut vide (UHV), appel DUF ( Dinamo UHV Factory). [1] Afin de transformer le TSQ700 en source dions basse nergie, nous avons simul le dernier quadruple laide du logiciel SIMION. Ltude de lnergie des ions a montr une traine haute nergie des ions : 41% des ions ont une nergie suprieure 40eV (Fig 1b Courbes bleues). Cette dispersion haute nergie est incompatible avec un dpt non destructif en soft landing (nergie des ions infrieure 5eV [2]). Pour trier les ions en nergie, nous avons ajout un triplet de lentilles lectrostatiques et un secteur lectrostatique (Fig 1a) [3]. La modlisation et loptimisation de ce dispositif a permis de dfinir les gomtries du triplet de lentilles et du secteur lectrostatique et doptimiser les tensions appliques sur ces lments pour supprimer la traine en nergie et ne garder que les ions arrivant avec une nergie infrieure 5 eV sur la palette (Fig 1b Courbes rouges). La contrepartie est que seulement 9% des ions entrants arrivent sur la palette. A partir du modle optimis du dispositif de filtre, le bureau dtude du CEMES a conu les pices pour les intgrer au TSQ700. Le secteur lectrostatique est mont sur un support qui reoit galement le porte-substrat surmont dun four permettant de chauffer lchantillon pour limiter le collage de molcules non ionises. Sous le secteur lectrostatique est install un dtecteur grilles (Fig 1a) pour mesurer lnergie des ions afin dajuster les polarisations des lectrodes du secteur lectrostatique avant le dpt. Un translateur permet de positionner lensemble secteur lectrostatique-palette-four-dtecteur en sortie de triplet de lentilles avec prcision pour caractriser les ions ou les dposer. Lensemble peut aussi tre relev afin dutiliser le TSQ700 en mode normal de fonctionnement. [1] http://www.duf.cemes.fr/ [2] S. Rauschenbach, R.Vogelgesang, ACS Nano, (2009), 3 (10), pp 29012910. [3] A. Pernot, M. Abignoli, M. Barat, Revue de Physique Applique, (1967), tome 2, pp 203-212. Link: http://www.cemes.fr/
169/381
[P83] Analyse lipidomique de foie gras de canard de diffrents classements commerciaux laide de limagerie par spectromtrie de masse TOF-SIMS.
Christopher Cerruti1, Laetitia Thron2, David Touboul1, Thierry Astruc3, Alain Brunelle1
1
1Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette
2
2INRA, INPT - ENSAT, INPT - ENVT, UMR 1289 Tissus Animaux, Nutrition, Digestion, cosystme et Mtabolisme, Avenue de l'Agrobiopole, 31326 Castanet Tolosan
3
INRA de Clermont Ferrand Theix, UR370 QuaPA, , 63122 St Gens Champanelle
Contact: Christopher Cerruti (christopher.cerruti@icsn.cnrs-gif.fr)
Keywords: Imaging mass spectrometry La production de foie gras partir de palmipdes est une activit conomique et traditionnelle de lagriculture franaise depuis cinq sicles. Le foie gras est le rsultat dun processus daccumulation de lipides pendant la priode dengraissement. Lors du traitement thermique, une partie des lipides accumuls pendant la phase de gavage peut migrer de la structure cellulaire vers la surface du foie : il s'agit du phnomne de fonte qui est exprim par le rendement de cuisson. Diffrentes techniques analytiques ont t utilises pour tenter dexpliquer la capacit dun foie gras conserver ou perdre sa matire grasse pendant la cuisson mais aucune dentre elles na permis didentifier ce qui prdfinit un foie ce phnomne. Les techniques dimagerie bases sur la spectromtrie de masse, comme la spectromtrie de masse ionisation secondaire par temps de vol (TOF-SIMS) permettent la cartographie de tout compos prsent la surface dune coupe de tissu avec une rsolution de lordre du micromtre [1]. Afin de dterminer la composition en lipide du foie gras, nous avons effectu une analyse lipidomique in situ en nous concentrant sur les gouttelettes lipidiques (remarque : les goutelettes lipidiques sont ici incluses dans les hpatocytes et non les adipocytes) et les membranes plasmiques des cellules hpatiques. Lobjectif de notre tude tait dvaluer la composition et la rpartition locale des lipides directement sur des coupes de foie de canard afin didentifier une diffrence qui puisse expliquer leurs caractristiques, comme cela fut fait rcemment avec des foies humains statosiques [2]. Les chantillons de foie gras analyss sont diffrenciables par le rendement post-traitement thermique ou par le classement commercial (extra, slection ou tout venant). Des coupes de foie gras provenant de diffrents classements commerciaux ont t analyses laide dun spectromtre TOF-SIMS IV (Ion-TOF GmbH, Mnster, Allemagne) quip dune source dagrgats de bismuth, et compares aux rsultats obtenus avec un foie maigre servant de tmoin. Limagerie TOF-SIMS permet de diffrencier les foies gras classs parmi les extras et slections des tout venants. En effet ces derniers prsentent des gouttelettes lipidiques moins denses en diacylglycrols que dans les deux autres classes de foie gras. Les vsicules statosiques sont majoritairement composes de DAG C32:0 dans les foies gras et le foie maigre. Seule limagerie par spectromtrie de masse permet une telle quantification relative localise. Les changements dans la composition lipidique du foie associs la suralimentation induisent une augmentation dans la proportion entre le cholestrol membranaire et la phosphatidylcholine. 1. Touboul D, Laprvote O, Brunelle A, Curr. Opin. Chem. Biol. In press, DOI: 10.1016/j.cbpa.2011.04.017 2. Debois D, Bralet MP, Le Naour F, Brunelle A, Laprvote O, Anal Chem 2009, 81, 2823-2831.
170/381
[P84] Analyse de la dynamique de formation de complexes entre lanneau de processivit de la rplication bactrienne et des peptides inhibiteurs par spectromtrie de masse native.
Philippe Wolff1, Philippe Dumas3, Gilles Guichard2, Philippe Hammann1, Dominique Y. Burnouf3
1 2
Plateforme Protomique Strasbourg-Esplanade, FRC 1589, IBMC, 15 rue Ren Descartes, 67084 Strasbourg
Institut Europen de Chimie et Biologie, Universit de Bordeaux-CNRS UMR 5248, CBMN, 2, rue Robert Escarpit, 33607 Pessac
3
UPR 9002 Architecture et Ractivit de lARN, 15 rue Ren Descartes, 67084 Strasbourg
Contact: Philippe Wolff (p.wolff@ibmc-cnrs.unistra.fr)
Keywords: Signaling, interactomics and post-translational modifications Lanneau de processivit est un composant essentiel du complexe rplicatif bactrien. Cet homodimre interagit avec de multiples protines impliques dans les processus du mtabolisme de lADN, comme la rplication et la rparation des lsions. Cette interaction est ncessaire afin que les protines puissent remplir leur fonction physiologique. Des comptiteurs de ces interactions ADN polymrases / anneau sont aptes provoquer larrt de la rplication et la mort cellulaire, dfinissant ainsi l'interface de liaison comme cible molculaire pour le dveloppement de nouveaux types dantibiotiques ciblant la rplication bactrienne. La structure de co-cristaux de lanneau avec le peptide de liaison de la DNA polymrase Pol IV dEscherichia coli (Burnouf et al. 2004) a dfini la poche dinteraction. A partir de ces donnes structurales, nous dveloppons des ligands synthtiques liant cette poche hydrophobe dans le but de dvelopper de nouvelles molcules synthtiques inhibitrices de la rplication bactrienne. Lanalyse de la dynamique de formation des complexes peptides / anneaux a t tudie par spectromtrie de masse native en fonction de la concentration en peptide. Les donnes exprimentales se superposent au modle thorique et les valeurs de Kd mesures par cette technique sont identiques celles mesures indpendamment par ITC et SPR et permettent une double validation de la spectromtrie de masse native pour la dtermination de constantes daffinit. Link: http://www-ibmc/proteo/accueil_029.htm
171/381
[P85] Uncovering the Cell Sialo-proteome Using Metabolic Oligosaccharide Engineering and Mass Spectrometry
Violette Gautier1, Nicolas Delcourt3, Emmanuelle Mouton-Barbosa1, Mathilde Beau1, Jean-Philippe Girard1, Odile Burlet-Schiltz1, Anne Gonzalez de Peredo1, Bernard Monsarrat1
1
Institut de Pharmacologie et de Biologie Structurale/CNRS, UMR5089, 205 route de Narbonne, 31077 Toulouse, France
2 3
Universit de Toulouse, 118 route de Narbonne, 31062 Toulouse, France Cancer Research Center of Toulouse, INSERM U1037 , 20-24 rue du pont saint-pierre, 31052 Toulouse, France
Contact: Violette Gautier (violette.gautier@ipbs.fr)
Keywords: Signaling, interactomics and post-translational modifications, Proteins, peptides and small molecules quantification N-glycosylation is among the most common post-translational modifications in cells and it is prevalent in proteins localized both on the extracellular side of the plasma membrane and in the extracellular space. Most of these glycoproteins contain sialic acid as the monosaccharide located on the non-reducing end of the glycans. Methods for global N-glycopeptides characterization have been described (1, 2), but they are not specific for the enrichment and the global analysis of membrane sialylated proteins. We present a strategy based-on metabolic oligosaccharide engineering (3) in combination with label free quantitative mass spectrometry for the specific detection and quantitation of the cell sialo-proteome dynamics. This method is based-on the metabolic incorporation of N-acetyl mannosamine azido to tag cell surface and secreted glycoproteins. This azido-sugar was further reacted very specifically with a phosphine-biotin probe, via a Staudinger ligation. Biotinylated glycoproteins were then purified and sequentially digested with trypsin and PNGase F. PNGase F digestion was performed in 18-O water, which allowed us to discriminate, thanks to different mass increments, deglycosylation of glycopeptides from spontaneous deamidation. Finally trypsin and PNGase F digested peptides were analyzed on an LTQ-Orbitrap Velos mass spectrometer. This strategy was applied for the in-depth characterization of the sialo-proteome in endothelial cells and to quantify its variation in response to pro-inflammatory treatments. We identified more than four hundred N-glycosylation sites corresponding to 202 sialylated glycoproteins. Using quantitative analyses, we also show that pro-infammatory cytokines leads to the upregulation of above 50 sialo-proteins involved in various pathophysiological functions such as vascular endothelial cell migration and adhesion. This study shows that proteomic analysis based on metabolic incorporation of azido-N-acetylmannosamine coupled with label-free quantitative MS measurements represents a powerful new approach to analyze global cellular sialo-proteome changes. Link: http://proteomique.ipbs.fr/
172/381
[P86] Sequencing lys-n proteolytic peptides by esi and maldi tandem mass spectrometry
Mathieu Dupr1, Galle Fromenty1, Sonia Cantel1, Jean Martinez1, Christine Enjalbal1
1
Institut des Biomolcules Max Mousseron (IBMM UMR 5247), Universit Montpellier 2, 34095 Montpellier
Contact: Mathieu Dupr (mathieu.dupre@univ-montp2.fr)
Keywords: Organic and inorganic mass spectrometry In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola Frondosa[1] that was recently investigated in proteomic studies[2] as an alternative to trypsin digestion since a specific cleavage at the amide X-Lys chain is obtained providing N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residues solely at the C-terminal position [3], [4], and thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with ESI-QqTof and MALDI-Tof/Tof mass spectrometers commonly used in proteomic bottom-up experiments were investigated [5]. [1] Nonaka, T.; Hashimoto, Y.; Takio, K., J. Biochem. 1998, 124, 157-162. [2] Coussot G, Hawke DH et al,. Anal Biochem. 2007; 361(2):302-304. [3] Boersema, P. J.; Taouatas et al., Moll. Cell. proteom. 2009, 8, 650-660. [4] Carabetta, V. J.; Li, T.; Shakya, A.; Todd, M. G., Cristea, I. M., J. Am. Soc. Mass Spectrom. 2010, 21, 1050-60. [5] Dupr, M.; Cantel S.; Verdi P.; Martinez J.; Enjalbal C., J. Am. Soc. Mass Spectrom. 2011, 22, 265-279
173/381
[P87] Etude protomique du secrtome chez Clostridium difficile

Cline Viala1, Sverine Pchin1, Anne Collignon1, Cline Boursier2
1
EA 4043, USC INRA, Facult de Pharmacie, Universit Paris-Sud 11, 5 rue JB Clment, 92296 ChtenayMalabry , France
2
Plate-Forme TransProt Activit Protomique, IFR141-IPSIT, Facult de Pharmacie, Universit Paris-Sud 11, 5 rue JB Clment, 92296 Chtenay-Malabry , France Contact: Cline Boursier (celine.boursier@u-psud.fr)
Keywords: Clinical proteomics Clostridium difficile est un pathogne responsable de diarrhes et de colites pseudomembraneuses, survenant le plus souvent aprs un traitement antibiotique. Les facteurs de virulence majeurs de Clostridium difficile sont les toxines A et B. Cependant, la colonisation du tube digestif, considre comme la premire tape de la pathognse, implique dautres protines de virulence dont certaines ont dj t caractrises. L'objectif de cette tude est de mettre en vidence de nouvelles protines qui pourraient tre impliques dans cette tape de colonisation. Par une approche protomique combinant l'lectrophorse bidimensionnelle (2DE) et la spectromtrie de masse, nous avons tudi le secrtome de C. difficile sur une cintique de croissance in vitro sur une souche virulente de C. difficile. Les 2DE ont t ralises partir d'extraits de protines secrtes prleves dans le surnageant de culture diffrents temps et prcipites. Aprs analyse d'images comparative des gels, 202 spots protiques diffrentiels ont t mis en vidence. Les premires analyses par nanoLC-MS/MS permettent d'identifier les principales protines scrtes. Link: http://ifr141.cep.u-psud.fr/transcriptome-proteome/index.html
174/381
[P88] Analysis of chlorinated compounds in tap water using PTR-ToF Mass Spectrometry: H3O+ versus O2+ chemical ionisation
Xavira Pennanec1, Etienne Smon1, Nolle Bno1, Thierry Thomas-Danguin1, Jean-Luc Le Qur1
1
Centre des Sciences du Got et de l'Alimentation, UMR6265 CNRS, UMR1324 INRA, Universit de Bourgogne, Agrosup Dijon, 17 rue Sully, 21000 Dijon Contact: Xavira Pennanec (Xaviera.Pennanec@dijon.inra.fr)
Keywords: Instrumentation, Organic and inorganic mass spectrometry For a safe human consumption, tap water needs to go through chemical, physical and biological treatments. Amongst the chemical treatments, chlorination is often used to minimize bacteriological contamination risks. However many consumers complain about bad taste or off-flavour in tap water which could be potentially induced by the chlorination process. Hypochlorous acid (HOCl) and chloramines are possible candidates for a chlorine off-odour. Chloramines may be byproducts from the reaction of chlorine with nitrogenous compounds that can be present all over the distribution network of tap water. The aim of this study was to detect the potential presence of chloramines and/or HOCl in tap water headspace using Proton Transfer Reaction Time of Flight Mass Spectrometry (PTR-ToFMS) and to compare the classical chemical ionisation with H3O+ to the chemical ionisation using O2+ using a switchable reagent ions (SRI) source to track these chlorinated compounds. In most of the PTR experiments, analyses are achieved using H3O+ as precursor ion, mainly leading to [MH]+ protonated ions. Ionisation is only possible for molecules with a proton affinity (PA) superior to that of water (PAWater = 691 kJ/mol). The molecules with a lower proton affinity than water can therefore not be ionized via proton transfer. The switchable seagent ions (SRI) system on the PTR-MS source (Ionicon Analytik, Innsbruck) enables to switch to another reactant ion than H3O+ like O2+. Charge transfer ionisation is thus possible with molecules with a lower ionisation potential than that of O2 (IPO2 = 12.06 eV), thus increasing the range of molecules which can be analysed. Headspace analyses were conducted using H3O+ and O2+ (to compare these two reagent ions). Ionisation parameters optimisation was especially focussed on H2O or O2 flows, pressure into the drift tube and E/N ratio (where E: electric field strength in the drift tube; N: buffer gas number density in the drift tube). The detected compounds were mainly hypochlorous acid (HOCl) and mono-, di- and trichloramines (ClNH2, Cl2NH and Cl3N respectively). The Time of Flight (ToF) mass analyser allowed the determination of exact mass of the different ions and thus the identification of analytes with certainty. Sensitivity of hypochlorous acid was greater towards O2+ reagent ion than H3O+. Dichloramine could not be identified by H3O+ chemical ionisation because of the presence of an intense mass peak of an unidentified contaminant (m/z = 85.93) close to the m/z of the dichloramine. On the other hand, dichloramine could be unambiguously detected using O2+ as the precursor ion thanks to its selectivity.
175/381
[P89] Utilisation de la source APCI en introduction directe pour lanalyse en matrices complexes
Florian Albrieux1, Christian Duchamp1, Natali Henriques1
1
Institut de Chimie Biochimie Molculaire et Supramolculaires UMR5246, Centre Commun de Spectromtrie de Masse, Bt. CPE, aile Curien, Campus de la Doua, 43 Bd du 11 nov. 1918, 69622 Villeurabanne CEDEX Contact: Florian Albrieux (florian.albrieux@univ-lyon1.fr)
Keywords: Instrumentation, Organic and inorganic mass spectrometry Cette tude prsente des exemples danalyses de composs prsents dans des matrices complexes en utilisant lionisation chimique pression atmosphrique en introduction directe. Les analyses sont ralises sans aucune prparation dchantillon. Nous prsenterons des exemples danalyse de composs prsents dans des urines ainsi que dans des milieux ractionnels comportant des solvants peu compatible avec lionisation electrospray (DMSO et THF).
176/381
[P90] Etude diffrentielle des protomes de branchies de dreissnes (moules deau douce) rcoltes sur des sites pollus ou non.
Bertrand NAUDIN2, Philippe CHAN TCHI SONG 3, Pascal COSETTE 2, Thierry JOUENNE 2, Marc PARANT 1, Sandrine PAIN-DEVIN 1
1
CNRS UMR 7146 Laboratoire Liebe (Interactions Ecotoxicologie, Biodiversit, Ecosystmes) , Universit Paul Verlaine - rue du Gnral Dlestraint, 57070 Metz
2
CNRS UMR 6270 Laboratoire PBS (Polymres, Biopolymres, Surfaces), Universit de ROUEN - Bd Maurice de Broglie, 76820 Mont Saint Aignan
3
INSERM U 982 Laboratoire Diffrenciation et Communication Neuronale et Neuroendocrine, Universit de ROUEN - Place Emile Blondel, 76820 Mont Saint Aignan Contact: Bertrand NAUDIN (bertrand.naudin@univ-rouen.fr)
Keywords: Proteins, peptides and small molecules quantification Des dreissnes ont t rcoltes dans huit sites de diffrentes rivires de France prsentant des degrs de pollution varis. Leurs branchies ont t dissques puis une extraction des protines a t effectue afin dtudier le protome de ces organes qui filtrent leau en permanence et qui pourraient servir de marqueurs de susceptibilit aux stress anthropiques. Les protines ont t spares par lectrophorse 2-D. Pour chaque site, quatre chantillons diffrents de dreissnes ont t traits, soit quatre gels par site. Les 32 gels obtenus ont t numriss puis une analyse diffrentielle a t entreprise en utilisant le logiciel SameSpots (Nonlinear Dynamics). Lanalyse en composante principale a fait ressortir deux groupes dchantillons dont les spots dintrt sont exprims diffremment. Le premier groupe rassemble les gels dchantillons de dreissnes issues des quatre sites qui sont parmi les plus pollus, tandis que le second rassemble les chantillons issus des autres sites, peu ou pas pollus. En vue de leur identification, ces spots ont t dcoups et digrs par la trypsine afin de les soumettre une spectromtrie de masse de type ESI-Quad-Tof. Les spectres obtenus ont t traits par le logiciel Mass Hunter (Agilent). Pour lidentification des protines, nous avons utilis MASCOT ( partir de la base de donnes NCBI ; taxonomy : other Metazoa), ainsi que des outils de recherches de squenage de novo avec MSBlast et PEAKS/SPIDER (Bioinformatics Solutions). Parmi les protines sous-exprimes en milieux pollus, on peut citer : proteasome 26S ATPase subunit, annexin, GADPH, triophosphate isomerase, histone H2B. Ces expressions diffrentielles de protines reprsentent sans doute chez les organismes qui les expriment des mcanismes de dfense qui leur permettent de rsister la prsence et limpact toxique des polluants d'origine anthropique. Cette tude a t faite dans le cadre du Programme SYDEPOP : INSU-SIC-EC2CO-Cytrix.
177/381
[P91] Etude de la voie de scrtion SecA2-dpendante chez Listeria monocytogenes par lanalyse de lexoprotome.
Sandra RENIER1, Ingrid CHAFSEY1, Christophe CHAMBON2, Michel HEBRAUD1, Mickal DESVAUX1
1
UR454 Microbiologie, Equipe QuaSA, INRA de Clermont-Ferrand, site de Theix, 63122 Saint-Gens Champanelle
2
Plate-Forme d'Exploration du Mtabolisme, composante protomique, INRA de Clermont-Ferrand, site de Theix, 63122 Saint-Gens Champanelle Contact: Michel HEBRAUD (michel.hebraud@clermont.inra.fr)
Keywords: Systems biology Listeria monocytogenes est lagent tiologique de la listriose, une infection grave dorigine alimentaire. Ce pathogne possde la capacit de survivre dans divers environnements comme le sol, les matrices alimentaires ou encore le cytosol des cellules eucaryotes. Les facteurs de virulence scrts, localiss dans lenveloppe cellulaire ou relargus dans le milieu extracellulaire, sont rguls par la temprature et sont essentiellement exprims 37C. Une large majorit des exoprotines sont prdites comme tant scrtes par la voie Sec. Le translocon Sec est compos dun htrodimre SecYEG formant un canal travers la membrane cytoplasmique, et de lATPase SecA. Plusieurs bactries Gram positif, dont L. monocytogenes, prsentent un paralogue de SecA, nomm SecA2. Contrairement la protine SecA qui est essentielle, labsence de SecA2 entrane uniquement un dfaut au niveau de la morphologie de la bactrie. Des analyses prliminaires ont rvl labsence ou la rduction significative dexoprotines chez le mutant dpourvu du gne secA2. De plus, certaines dentre elles sont dpourvues de peptides signal et par consquent ne peuvent pas tre prdites comme tant scrtes par le systme Sec. Le but de notre tude est de caractriser lexoprotome du mutant secA2 chez L. monocytogenes. Les protines prsentes dans le milieu extracellulaire ont t isoles et rsolues en lectrophorse bidimensionnelle Suite leur identification en spectromtrie de masse MALDI-TOF, le sous-ensemble de protines extracellulaires scrtes d'une manire SecA2-dpendante a pu tre tabli chez L. monocytogenes la fois 20C et 37C. Paralllement, une approche protomique hors-gel par LC-MS/MS (LTQ Velos) a t effectue sur la souche sauvage. Outre l'expression diffrentielle des protines, la fonction des exoprotines SecA2-dpendantes possdant ou non un peptide signal sera discute.
178/381
[P92] Structural analysis of synthetic polymers using MALDI SpiralTOF-TOF with high-energy CID
Takaya Satoh1, Ayumi Kubo1, Yoshiyuki Ito1, Masahiro Hashimoto1, Masaaki Ubukata2, Takafumi Sato1, Jun Tamura1, Akihiko Kusai3
1 2 3
JEOL Ltd., 1-2, Musashino 3-Chome Akishima, 196-8558 Tokyo, Japan JEOL USA, Inc., 11 Dearborn Rd., 01960 Peabody, MA USA JEOL (EUROPE) SAS, Espace Claude Monet - 1, Alle de Giverny, 78290 Croissy-sur-Seine, France
Contact: Akihiko Kusai (kusai@jeol.fr)
Keywords: High mass and high resolution mass Spectrometry, Organic and inorganic mass spectrometry, Instrumentation Recently, a tandem time-of-flight mass spectrometer (TOF-TOF) with matrix-assisted laser desorption/ionization (MALDI) is used for structural analysis of synthetic polymer. A high-energy collision-induced dissociation (HE-CID) is mostly used method to make a fragmentation. There are two factors which make interpretation of a HE-CID product ion spectrum difficult: i) overlap of fragment pathways of HE-CID and post-source decay (PSD) on a product ion spectrum, ii) insufficient separation of fragment peaks caused by low precursor ion selectivity and low mass resolution. The MALDI TOF-TOF combined a SpiralTOF [1] and an offset parabolic reflectron for first and second TOFMS, respectively has been developed. The SpiralTOF, which has 17 m flight path, can select a monoisotopic ion so that each fragment pathway is observed as single peak on product ion spectrum. Furthermore, PSD ions are eliminated by electrostatic sectors inside the SpiralTOF. As a result, fragment pathways using HE-CID are preferentially observed on product ion spectrum. In this study, some synthetic polymers were investigated for structural analysis using HE-CID spectrum acquired by MALDI SpiralTOF-TOF. A repeat structure of synthetic polymer was clearly observed on a product ion spectrum of selected monoisotopic precursor ion of [M+Na]+. At the same time, a fragment pathway related to near end-group was observed as single peaks in a low m/z region. [1] Satoh, T.; Sato, T.; Tamura, J. J. Am. Soc. Mass Spectrom. 2007, 18, 1318. Link: www.jeol.com
179/381
[P93] Ciblage par MALDI-TOF-MS de nouveaux composs inhibiteurs des protines phosphatases CDC25s
Estelle Sibille1, Emilie Bana2, Denyse Bagrel2, Gilbert Kirsch2, Patrick Chaimbault2
1 2
Laboratoire de Spectromtrie de Masse et Chimie Laser, ICPM 1, bd Arago, 57078 Metz Cedex 03, France
Laboratoire d'Ingnierie Molculaire et Biologie Pharmacologique, ICPM 1, bd Arago, 57078 Metz Cedex 03, France Contact: Estelle Sibille (estelle.sibille@univ-metz.fr)
Keywords: High mass and high resolution mass Spectrometry, Organic and inorganic mass spectrometry Les phosphatases CDC25s, qui comportent trois isoformes A, B et C, jouent un rle majeur dans le processus cancreux plusieurs niveaux du cycle cellulaire. Actuellement, les recherches se focalisent sur linhibition de ces protines grce des molcules naturelles ou synthtiques. Ces molcules inhibent laction de ces protines en bloquant rversiblement (ex : indolylhydroxyquinones [1]) ou irrversiblement (BN82002 [2]) leur site actif, empchant ainsi le bon droulement du cycle. Le travail a consist dvelopper un protocole permettant de cribler des inhibiteurs de CDC25s. Ce protocole volue selon la rversibilit ou lirrversibilit de la liaison inhibiteur-site catalytique de la protine. La technique de criblage mise au point prcdemment au laboratoire [3] permet dj de dtecter par MALDI-TOFMS le signal dun inhibiteur non covalent (rversible). Cependant, il ne peut sappliquer au criblage dinhibiteurs covalents (irrversibles). Afin de pouvoir cribler ces derniers, nous avons complt le protocole par une approche Peptide Mass Fingerprinting permettant la dtection systmatique du site actif des CDC25s. Les spectres de masse obtenus par MALDI-TOF (ou MALDI-FTICR) sont soumis la banque de donnes MASCOT afin didentifier les peptides issus de la digestion trypsique. Cette approche est applique aux CDC25s humaines (produites et purifies au laboratoire) seules et mises en contact avec les candidats inhibiteurs. Les diffrences entre protines inhibes ou non correspondent la dtection dun pic correspondant au site actif de la protine dcal de la masse de linhibiteur et/ou la disparition du pic correspondant leur site catalytique. Par exemple, aprs digestion, le site catalytique des CDC25s nest systmatiquement plus identifi par MASCOT lorsque le BN82002 est soumis ce test. Ce test permet donc de diffrencier pour les CDC25s, les inhibiteurs covalents des inhibiteurs non covalents. Ce protocole permet de slectionner avec discernement les molcules candidates aux futurs tests in cellulo avec les CDC25s. Le caractre covalent de la liaison rvl par ce protocole permet de slectionner plus finement les composs dintrt car les inhibiteurs irrversibles permettent de bloquer dfinitivement leur site actif. Rfrences : [1] J. Sohn et al. Inhibition of Cdc25 Phosphatases by Indolyldihydroxyquinones, J. Med. Chem. 46 (2003) 2580-2588. [2] M.C. Brezak et al. A Novel Synthetic Inhibitor of CDC25 Phosphatases : BN82002, Cancer Res. 64 (2004) 3320-3325. [3] P. Hannewald et al. Tubulin-Binding Drug Screening by MALDI-TOFMS, Anal. Chem. 78 (2006) 4390-4397.
180/381
[P94] Mise au point dune mthode de dtection et de quantification multiplexe par MRM des peptides amylodes dans le LCR humain
Pauline Poinot1, Christophe Hirtz2, Audrey Gabelle3, Laurent Tiers2, Christophe Junot1, Sylvain Lehmann1, Franois Becher1
1 2
CEA, iBiTec-S, Service de Pharmacologie et dImmunoanalyse, Saclay, 91191 Gif-sur-Yvette, France
CHRU de Montpellier, Biochimie - Protomique Clinique, IRB-Hpital St Eloi, 80 av A. Fliche, 34295 Montpellier, France
3
CHRU de Montpellier, Service de Neurologie, Hpital Gui de Chauliac, 80 av A. Fliche, 34295 Montpellier, France Contact: Franois Becher (francois.becher@cea.fr)
Keywords: Proteins, peptides and small molecules quantification , Separative analysis methods Comptant plus de 860000 personnes atteintes en France, la maladie dAlzheimer est devenue aujourdhui un vritable problme de sant publique. A ce jour, le diagnostic de la maladie repose essentiellement sur un examen clinique, de limagerie, et des tests neuro-psychologiques. Afin de complter ce diagnostic, une nouvelle approche, lidentification de protines marqueurs dans les biofluides, a merg. Les peptides amylodes, ainsi que la protine Tau, constituent des marqueurs dintrt pour le diagnostic de la maladie. Chez les sujets atteints, leur agrgation entrane la formation de plaques sniles extracellulaires contribuant une dysfonction et une dgnration des neurones. Parmi ceux-ci, les peptides A 40 et 42 semblent tre des marqueurs potentiels de la maladie dAlzheimer (Graff-Radford et al., 2007). En outre, dautres formes ont t identifies dans le LCR, et pourraient galement tre lies lvolution de la maladie (Portelius et al., 2009). Jusqu prsent, ces marqueurs ont essentiellement t dtects et valids au travers des techniques immunologiques. Or, le cot et le temps ncessaires lobtention danticorps spcifiques, de mme que la difficult de multiplexer ces mthodes nen font pas toujours des techniques de choix, notamment en vue dune application clinique. La spectromtrie de masse semble tre aujourdhui une bonne alternative (Portelius et al., 2007). Au cours de cette tude, nous avons mis eu point une mthode de ciblage et de dosage multiplex de peptides A par spectromtrie de masse en tandem. Diffrents LCR de patients ont t analyss par LC/MSMS en mode MRM. Pour chacun des peptides cibls, 4 6 transitions ont t dtermines. La quantification absolue des peptides A 40 et 42 a t ralise via lutilisation de standards marqus. Les rsultats obtenus rvlent quune analyse LC/MSMS permet de dtecter de faon sensible lensemble des peptides A cibls. De plus, cette approche haut dbit danalyse permet lanalyse dchantillons issus de larges cohortes de patients. Aprs validation clinique, elle pourrait lavenir tre utilise pour le diagnostic de la maladie dAlzheimer.
- Graff-Radford NR, Crook JE, Lucas J, Boeve BF, Knopman DS, Ivnik RJ, Smith GE, Younkin LH, Petersen RC, Younkin SG (2007). Arch Neurol 64(3), 354-62. - Portelius E, Brinkmalm G, Tran AJ, Zetterberg H, Westman-Brinkmalm A, Blennow K. (2009). Neurodegener Dis. 6(3):87-94. - Portelius E, Tran AJ, Andreasson U, Persson R, Brinkmalm G, Zetterberg H, Blennow K, Westman-Brinkmalm A. (2007). J Proteome Res. 6(11):4433-9.
181/381
[P95] Synthse et valuation de deux sondes chimiques spcifiques pour le marquage et lanalyse protomique des glycoprotines des cellules endothliales humaines au cours de la rponse inflammatoire
Violette Gautier1, Mathilde Beau1, Michel Azoulay2, Laura Lano2, Nicolas Delcourt1, Anne Gonzalez de Peredo1, Jean-Claude Florent2, Odile Burlet-Schiltz1, Bernard Monsarrat1
1
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, 205 route de Narbonne, 31077 Toulouse
2
Institut Curie (Conception, synthse et vectorisation de biomolcules) UMR 176, 26 rue dULM, 75248 Paris cedex 05 Contact: Violette Gautier (violette.gautier@ipbs.fr)
Keywords: Organic and inorganic mass spectrometry, Proteins, peptides and small molecules quantification Le glycoprotome de la surface cellulaire est impliqu dans des processus biologiques fondamentaux tels que la transduction du signal, le transport de molcules, le trafic membranaire, la migration des cellules et les interactions intercellulaires. Cependant, il est galement lun des plus difficile analyser, car les protines impliques sont trs hydrophobes, difficiles solubiliser, purifier, et ne reprsentent quune fraction mineure du contenu protique total de la cellule. Actuellement, lune des approches utilises pour lidentification des glycoprotines de surface repose sur une stratgie de marquage bioorthogonale1,2. Cette mthode est ralise en deux tapes : incorporation mtabolique et marquage chimique. Dans un premier temps, une fonction reportrice de type azoture est introduite au niveau des glycannes via la machinerie cellulaire, par ajout dans le milieu de culture dun prcurseur monosaccharidique modifi. Dans un second temps, cette fonction reportrice se lie de faon covalente une sonde exogne grce une fonction complmentaire de type triarylphosphine ou alcyne. Dans la littrature, seules les ractions de Staudinger2 et de click chemistry sans cuivre3 permettent in vivo le marquage chimique des azido-glycoprotines intermdiaires. Cette tude prsente dune part la synthse chimique de deux sondes possdant une biotine lie un groupement triarylphosphine4 ou monofluorocyclooctyne5, et dautre part lvaluation compare des performances de ces deux sondes pour le marquage et lanalyse protomique des glycoprotines. Les rsultats obtenus indiquent que la phosphine-biotine, bien quextrmement spcifique, prsente une moindre efficacit de marquage et soxyde rapidement. La fluoro-cyclooctyne teste prsente en revanche une vitesse de raction bien suprieure celle de la phosphine, permettant denvisager le suivi de processus cellulaires rapides. Cette stratgie, combine une analyse protomique quantitative sans marquage grce au logiciel MFPaQ, a ainsi permis de raliser une tude cintique de la dynamique du sialo-glycoprotome des cellules endothliales humaines au cours de la rponse inflammatoire. Link: http://www.ipbs.fr/
182/381
[P96] Comparison of different liquid chromatography systems for the detection of low-level biomarkers in biological fluids by Selected Reaction Monitoring
Florence Roux-Dalvai1, Violette Gautier1, Alexandre Stella1, Jrme Lemoine2, Anne Gonzalez de Peredo1, Bernard Monsarrat1, Odile Burlet-Schiltz1
1
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, Universit de Toulouse, 205, route de Narbonne, 31077 Toulouse, France
2
Institut des Sciences Analytiques, UMR 5280 CNRS, Universit de Lyon, -, 69622 Villeurbanne, France
Contact: Florence Roux-Dalvai (florence.dalvai@ipbs.fr)
Keywords: Proteins, peptides and small molecules quantification , Clinical proteomics, Separative analysis methods Detection of low-abundant proteins in biological samples is usually hampered by limited sensitivity and dynamic range of mass spectrometers. In the Selected Reaction Monitoring (SRM) mode, the dynamic range problem is alleviated, as target parent and fragment ions are respectively selected in a narrow mass range window by the two quadrupole analyzers, filtering out other highly abundant peptide ions that otherwise would lead to signal suppression in a full MS scan. Nonetheless, SRM analysis of weakly concentrated molecule results in low ion currents in the mass spectrometer, and intrinsic sensitivity of the instrument may then become the limiting factor of the analysis. Injecting higher amounts of analyte in the mass spectrometer could thus in this case help to improve the detection of species present at very low level in a complex matrix. Here, we tested different sample processing methods and LC-MS instrumental settings to optimize the detection of biomarker candidates in two biological fluids. Increasing amounts of sample were loaded either on a 75m I.D. capillary column coupled to a nanospray source, or on a 2mm I.D. column coupled to an ESI source. In each case, several proteins were monitored by SRM on a 5500QTrapTM mass spectrometer (ABSciex), in order to determine the optimal loading capacity of the chromatographic system. In the case of the nanoLC setting, samples were loaded on C18 precolumns with different loading capacity, and gradients of different lengths were tested in order to increase the amount of material loaded on the system without affecting the linearity of the MS response. For each experimental setting, the limit of detection by SRM of candidate biomarkers, spiked at different concentrations in plasma or cerebrospinal fluid, was determined. Our results indicate that the loss of sensitivity of conventional HPLC compared to nanoLC is balanced by the higher amount of sample that can be injected in the instrument, making this instrumental configuration an interesting alternative for the detection of low-abundant species, as long as the absolute quantity of starting material does not represent a limiting factor. Link: http://proteomique.ipbs.fr/
183/381
[P97] Silver interaction in the prion octarepeat domain

Bruno Bellina1, Isabelle Compagnon1, Rodolphe Antoine1, Michel Broyer1, Philippe Dugourd1, Alexander Kulesza2, Roland Mitric2, Vlasta Bonai-Kouteck2
1
Laboratoire de spctromtrie ionique et molculaire, CNRS LYON1, 43, Boulevard du 11 Novembre 1918, 69622 VILLEURBANNE , FRANCE
2
Department of Chemistry Humboldt-Universitt zu Berlin, Brook-Taylor-Str. 2, D-12489 BERLIN, ALLEMAGNE
Contact: Philippe Dugourd (dugourd@lasim.univ-lyon1.fr)
Keywords: Systems biology PRION, proteinaceous infectious particle, is an infectious agent composed of protein in a misfolded form. This new structure, which is influenced by metal binding, becomes pathological and is involved in many diseases such as Creutzfeldt-Jakob. The prion protein is known to binds metal in its N-terminal octarepeat domain. This domain is composed of tandem repeats of the sequence PHGGGWGQ. Recent work showed that each HGGGW (Histidine, Glycine and Tryptophan) segment may accommodate on metal ion. We aim at investigating the coordination of silver in this amino acid sequence and his influence on the peptide conformation. Using a mass spectrometer coupled with OPO lasers in the UV and IR regions, we measured the optical and vibrational spectra of the [HGGGW+Ag]+ complex and of several other sequences. Optical properties, binding site and peptide confirmation will be discussed. We will also report ion mobility spectroscopy and theoretical results.
184/381
[P98] Etude lipidomique de coupes de cerveaux post-mortem issus de patients atteints de la maladie dAlzheimer : apport des approches globales et semiglobales.
Mathieu Gaudin1, Ma Panchal4, Sophie Ayciriex1, Mlaine Courcoul1, Erwan Werner3, David Touboul2, Nicolas Auzeil1, Alain Brunelle2, Charles Duyckaerts 4, Olivier Laprvote1
1
Laboratoire de Chimie-Toxicologie Analytique et Cellulaire, Facult des Sciences Pharmaceutiques et Biologiques, Universit Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris Cedex 06
2
Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex
3 4
Division Mtabolisme, Technologie Servier, 25/27 rue Eugne Vignat, BP 1749, 45007 Orlans Cedex 1
Laboratoire de Neuropathologie Escourolle, Hpital de la Piti-Salptrire, 47/83 boulevard de l'hpital, 75013 Paris
5
Centre de recherche de l'ICM, 47/83 boulevard de l'hpital, 75013 Paris
Contact: Mathieu Gaudin (mathieu.gaudin@parisdescartes.fr) Keywords: Proteins, peptides and small molecules quantification , Separative analysis methods La maladie dAlzheimer (MA) se caractrise par la prsence de deux lsions crbrales, les plaques sniles et les dgnrescences neurofibrillaires. Les mcanismes physiopathologiques qui aboutissent leur constitution restent mal compris. A lheure actuelle, lallle epsilon-4 du gne codant lapolipoprotine E (ApoE), un transporteur des lipides, est considr comme lun des facteurs de risque le plus important de la maladie. Dautre part, lApoE a t identifie dans les plaques sniles. Ces donnes suggrent que des modifications dans le transport et dans le mtabolisme des lipides jouent un rle dans la pathognse de la MA. Afin dtablir le profil lipidique crbral post-mortem de patients atteints de la MA, nous prsentons une stratgie danalyse comprenant lextraction des lipides totaux partir de coupes de cerveau et leur identification par couplages chromatographie liquide - spectromtrie de masse suivant deux approches prsentant des degrs de slectivit croissants. Une approche dite globale utilise le couplage de la chromatographie liquide polarit de phase inverse afin de sparer les lipides par leur hydrophobicit. Leur dtection est assure par spectromtrie de masse haute rsolution temps de vol (Synapt G2 HDMS) permettant la dtermination des compositions lmentaires. Afin dobserver des modifications plus fines du lipidome, une approche semi-globale a t dveloppe et applique lidentification de six classes de phospholipides et de sphingolipides. Aprs sparation par chromatographie liquide polarit de phase inverse, les lipides sont slectionns par balayages spcifiques, ions prcurseurs et pertes de neutres, laide dun spectromtre de masse de type triple quadriple, afin de dtecter slectivement les lipides appartenant une classe dtermine. Une analyse statistique multivarie, ralise partir de profils lipidiques prcdents, a mis en vidence une diffrence de concentration dans certaines familles de lipides entre les patients atteints de la maladie dAlzheimer et des sujets non atteints. Lapproche globale indique notamment une augmentation des cramides dans lensemble du cortex temporal. Elle est confirme par une analyse semi-globale ciblant les cramides et pourrait tre due une hydrolyse des sulfatides. Nous avons galement observ une augmentation des sphingomylines mais uniquement dans la substance blanche. Sachant que par ailleurs Grimm et al [Nature Cell Bio, 2005] ont montr une augmentation de lactivit de la sphingomylinase dans les cultures de neurones exposes au peptide -amylode 1-42, notre rsultat semble suggrer lexistence dun mcanisme diffrent dans les fibres de myline et dans les corps cellulaires des neurones.
185/381
[P99] Towards quantitative analysis of sweat proteome

Anne-Marie Hesse1, Alexandra Kraut1, Damien Barthe1, Jean-Claude Launay2, Gustave Savourey2, Christophe Bruley1, Yohann Cout1
1 2
Biologie Grande Echelle, CEA / INSERM 1038 / UJF, 17 rue des Martyrs, 38054 Grenoble cedex 9, France
Ple Tolrance Climatique et Vtement CRSSA / DFH, , 24 Avenue des Maquis du Grsivaudan, 38702 La Tronche cedex, France Contact: Anne-Marie Hesse (anne-marie.hesse@cea.fr)
Keywords: Proteins, peptides and small molecules quantification , Bioinformatics et biostatistics dedicated to proteomics During interventions, emergency workers or servicemen must face extreme situations that may deeply impact their physiological behaviour. Real-time monitoring of biomarkers reflecting subject state would allow to prevent any medical accident by anticipating their pullback from the danger zone. In this context, sweat represents a biological fluid of choice for non-invasive detection of that kind of markers. We therefore undertook a proteomic analysis of human sweat obtained from donors submitted to various environmental constraints. We first explored the preparation of sweat proteins to bring them to liquid chromatography coupled to mass spectrometry analyses. Our results indicate that ultrafiltration combined with SDS-PAGE gave the best results in terms of protein recovery and number of identifications. To date, more than 300 proteins have been identified with high confidence in this biological fluid, which represents the biggest contribution to the establishment of a repertoire of human sweat proteins. In order to compare sweat proteome from subjects submitted to different constraints, isobaric tagging was chosen as the quantitative proteomics strategy. Several tests were conducted in terms of chemical label (TMT versus iTRAQ) or fragmentation mode (PQD versus HCD). Finally iTRAQ labelling was selected and adapted to in-gel protein digestion; labelled peptides were submitted to HCD which gave the more accurate results A great effort was consented to integrate the quantification data in the automated pipeline of identification validations and comparisons developed in-house (validated with IRMa, structured within Mass Spectrometry Identification databases MSIdb and analysed with hEIDI). The chosen strategy is currently applied on sweat samples collected from a cohort of 20 subjects submitted to 3 different environmental constraints: at rest in a hot area, at rest in a warm and humid atmosphere and with physical exercise in a warm and humid atmosphere .
186/381
[P100] Effets de lactylation des peptides de la peau de la rainette Pachymedusa dacnicolor pour un squenage de Novo par MALDI-TOF/TOF.
Claire Lacombe1, Constance Auvynet3, Germain Tong1, Grard Bolbach1, Emmanuelle sachon1
1 2 3
Universite P. et M. Curie Paris 6, UMR 7203 CNRS-UPMC-ENS, 4, place Jussieu, 75005 Paris, France Plateforme de spectromtrie de masse et protomique, IFR83, 7-9, quai saint Bernard, 75005 Paris, France Piti salptrire, 91, Bd de l'hpital, 75013 Paris, France
Contact: Emmanuelle sachon (emmanuelle.sachon@upmc.fr)
Keywords: Clinical proteomics Les grenouilles dAmrique latine synthtisent et scrtent au niveau de leur peau, des peptides biologiquement actifs. Dans cette tude, nous nous intressons aux peptides issus de la peau de la rainette, Pachymedusa dacnicolor, afin disoler et caractriser de nouveaux peptides dintrt thrapeutique. Pour ce faire, nous travaillons sur des exsudats de peau. Les peptides contenus dans ces exsudats sont spars par tamis molculaire suivi dHPLC. Les fractions obtenues sont testes pour leur activit biologique puis 4 fractions actives ont t analyses par spectromtrie de masse. Dans ces fractions, 6 peptides ont t identifis et fragments par MALDI-TOF/TOF (squenage de Novo). Deux dentre eux avaient dj t publis (DMS-DA5 et DMS-DA8). Le troisime correspond au peptide DMS-DA6 de la mme tude mais dont lextrmit C-terminale est sous forme acide carboxylique (GVWGIAKIAKIAGKVLGNILPHVFSSNQS-COOH). Les trois autres peptides coexistent dans la quatrime fraction et possdent une structure primaire Nterminale identique (rsidus 1 29). Cette squence commune, qui avait t dduite partir de l'ADN de grenouille en 1998 est la suivante : ALWKTLLKKVGKVAGKAVLNAVTNMANQN-COOH. Les deux autres peptides de cette fraction possdent ces 29 rsidus +E et +EQ en C-terminal. Le peptide DMS-DA6 a t synthtis sous forme amide ou non en C-terminal. Lactivit antimicrobienne des deux peptides a t teste sur deux souches de bactries (Gram+ et -) et leur structure secondaire dtermine par dichrosme circulaire. Tous ces peptides de peau de grenouille contiennent gnralement une grande quantit de rsidus K, il a donc t ncessaire de recourir une raction dactylation afin de lever lambigit Q/K qui existe du fait de la limitation dans la prcision de mesure de masse de linstrumentation MALDI-TOF (20 ppm pour les peptides). Lactylation des 4 fractions HPLC nous a permis de lever les ambiguts K/Q et nous a montr que cette raction peut avoir lieu sur les fonctions amine primaires (N-terminal et rsidus K) mais aussi, dans une moindre mesure, sur la fonction amine secondaire des rsidus H. Lactylation complte des peptides conduit la disparition des sites protonables (si aucun rsidu R dans la squence). Ceci entraine la disparition de la molcule monoprotone [M+H]+ au profit des espces [M+Na]+ et [M+K]+. Dans le cas de la prsence dun rsidu P dans la squence de ces peptides, on observe une fragmentation source, prfrentielle en N-terminal du rsidu P. Ces peptides actyls constituent de bons modles pour aborder le phnomne dionisation en MALDI. Link: http://www.chimie.ens.fr/LBM/
187/381
[P101] Differential proteomic analysis: iTRAQ vs zoomed 2D gel. Illustration by the study of Pseudomonas aeruginosa biofilms grown on glass wool.
Anne Marie Lomenech 1, Delphine Lapaillerie1, Jean William Dupuy1, Stephane Claverol1, Michel Perrot1, Marc Bonneu1, Sbastien Vilain1
1
Plateforme Protome Centre de Gnomique Fonctionnelle Bordeaux, 146 rue Lo Saignat, 33076 BORDEAUX Cedex Contact: Sbastien Vilain (sebastien.vilain@u-bordeaux2.fr)
Keywords: Proteins, peptides and small molecules quantification Bacterial biofilms are communities of bacteria attached to a support and embedded in a matrix of exopolymers. In this form of life, immobilized bacteria become hyper-resistant to all environmental stresses, particularly to antibiotics. Biofilms are therefore a major public health problem. Research conducted in many areas where these biofilms are found are intended to identify key genes involved in the establishment or maintenance of biofilms and / or resistance to antibiotics. As part of this research, we compared the protein content of P. aeruginosa grown as biofilm or suspended via two techniques of proteomic analysis: An off-gel approach, iTRAQ and an in-gel approach, zoomed 2D electrophoresis. We present here a quantitative and qualitative comparison of results obtained with these two techniques by highlighting the similarities and differences.
188/381
[P102] ICM-MS analysis to display specific protein interactions between cells (spermatozoa) and biologic fluid (seminal plasma)
Ana-Paula Teixeira-Gomes1, Laure Dewaele1, Guillaume Tsikis3, Nadine Grard3, Xavier Druart3, Valrie Labas1
1
INRA, Plate-forme dAnalyse Intgrative des Biomarqueurs, Centre de Recherches INRA de Tours, 37380 Nouzilly
2 3
INRA, UR1282, Infectiologie Animale et Sant Publique, Centre de Recherches INRA de Tours, 37380 Nouzilly
INRA, UMR 85, Physiologie de la Reproduction et des Comportements UMR CNRS 6175 Universit de Tours -Institut Franais du Cheval et de lEquitation, Centre de Recherches INRA de Tours, 37380 Nouzilly Contact: Ana-Paula Teixeira-Gomes (teixeira@tours.inra.fr)
Keywords: Systems biology Spermatozoa are produced by the testis. They need to transit through the epididymis to achieve their maturation and acquire functional and physiological competence to fertilize the oocyte. Intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) is a well established method to detect low molecular weight (<30kDa) compounds (peptidomic/proteomic). This fast and reliable method has been used first for identification of bacteria by comparison of MS fingerprints. It has recently been extended to the analysis of eukaryotic cells to display biomarkers. The spectra from whole cells reflect only a small part of the cellular protein. Indeed, ICM-MS promotes the desorption/ionization of the protein membrane or protein linked to the surface cell and becomes an original approach to study interaction between a fluid and the cells. In the present study, ICM-MS was used to visualize changes in spermatozoa protein patterns during their epididymal maturation. The goal was to discriminate seminal plasma proteins (originating from several glands) which could be involved in the final maturation of gametes, and may be linked to fertility. Epididymal (EPI) and ejaculated (EJAC) spermatozoa, as well as seminal plasma (SP), were collected from boars. Some EPI spermatozoa were incubated in the presence of saline or SP for 10 min at 37C. Then, EPI, EJAC and EPI+SP spermatozoa were mixed in a sinapinic acid matrix solution prior to generate MALDI-TOF profiles in the 2-20 kDa mass range. Differentially expressed peptides or proteins were found according to a probability value < 0.01 with the Progenesis MALDITM software (Nonlinear Dynamics). The comparison of the ICM-MS profiles of the three populations (EPI, EPI + SP, EJAC) showed protein modifications linked to interactions between the fluid and the cells. Among the 229 peaks detected, 11 were less intense in EPI+SP and EJAC groups, whereas 32 were detected with a larger intensity in EPI+SP and EJAC groups. Some of them may correspond to spermadhesins that are present in large amounts in seminal plasma. Of note is the fact that most modifications are still unknown. In conclusion, we demonstrated for the first time the suitability of the ICM-MS to study interactions of proteins from a biologic fluid (seminal plasma) with eukaryotic cells (spermatozoa). This approach may be adapted to identify newly molecular species involved in male fertility.
189/381
[P103] Etude de la conformation de peptides lasso par mobilit ionique et simulations REMD
Fabien Chirot1, Sverine Zirah3, Carlos Afonso4, Sylvie Rebuffat3, Philippe Dugourd2, Jean-Claude Tabet4
1
Institut des Sciences Analytiques, UMR 5280 CNRS-Universit Claude Bernard-Lyon1, 43 Boulevard du 11 Novembre 1918 , 69622 Villeurbanne, France
2
Laboratoire de Spectroscopie Ionique et Molculaire, UMR 5579 CNRS-Universit Claude Bernard-Lyon1, 43 Boulevard du 11 Novembre 1918 , 69622 Villeurbanne, France
3
Molcules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS-Musum National dHistoire Naturelle, 57 rue Cuvier, 75005 Paris, France
4
Institut Parisien de Chimie Molculaire, UMR 7201 CNRS-Universit Pierre et Marie Curie-Paris6, 4 place Jussieu, 75252 Paris, France Contact: Fabien Chirot (fabien.chirot@univ-lyon1.fr)
Keywords: Ionic mobility Les peptides lasso se caractrisent par une structure primaire particulire mettant en jeu une liaison covalente entre le groupement amine N-terminal et la chane latrale dun acide aspartique ou glutamique en position 8 ou 9. Leur structure secondaire forme un lasso o la chane principale passe travers la boucle ainsi cre pour former un nud. En raison de leurs proprits intressantes, notamment antimicrobiennes, lies leur structure en lasso, de nombreux efforts on t mis en uvre pour russir synthtiser de tels peptides initialement produits par des bactries. Leur structure particulire, extrmement contrainte, est de plus relativement unique dans le domaine des biomolcules. Afin de mieux comprendre les facteurs qui stabilisent cette structure en lasso, et notamment linfluence des interactions intramolculaires, nous avons tudi par mobilit ionique une srie de peptides modles drivs de la capistruine, peptide lasso de 19 acides amins produit par Bhurkholderia thailandensis E264. A travers la mesure de sections efficaces de diffusion dions dans un gaz, cette technique permet dune part de mettre en vidence lventuelle coexistence de diffrents conformres, et dautre part, via la comparaison avec des simulations, dobtenir des informations structurales. Nous avons donc ralis des simulations de dynamique molculaire par change de rpliques (REMD) bases sur le champ de forces AMBER99 afin dexplorer de manire efficace le paysage conformationnel complexe de ces peptides, et daider linterprtation des donnes exprimentales. Nous prsenterons les rsultats obtenus pour trois peptides modles ayant une squence identique mais une forme diffrente : la forme lasso (L), une forme comportant une boucle, mais pas de nud (S), et une forme linaire (C). Les rsultats exprimentaux montrent que, de manire assez surprenante, les trois formes prsentent des sections efficaces de diffusion voisines. De plus, les profils de mobilit mesurs rvlent la coexistence de plusieurs conformations pour les trois formes, y compris pour la forme L pourtant trs contrainte. Les simulations peinent reproduire quantitativement lexprience en raison de la taille des peptides, qui atteint les limites de loutil thorique. Cependant ces rsultats suggrent que lexistence de ponts salins pourrait tre lorigine de la compacit des structures observes pour la forme linaire en ajoutant une contrainte structurale similaire celle cause par lexistence dune boucle.
190/381
[P104] Couplage laser/mobilit ionique fragmentation rsolue en conformation

Lise Morlet1, Luke MacAleese2, Fabien Chirot1, Jrme Lemoine1, Philippe Dugourd2
1
2
Laboratoire de Spectroscopie Ionique et Molculaire, UMR 5579 CNRS-Universit Claude Bernard-Lyon1, 43 Boulevard du 11 Novembre 1918 , 69622 Villeurbanne, France Contact: Fabien Chirot (fabien.chirot@univ-lyon1.fr)
Keywords: Instrumentation, Ionic mobility La dtermination de la structure de systmes molculaires complexes tels que les protines ou les complexes protiques reprsente un enjeu important aussi bien pour la comprhension du fonctionnement de ces difices au niveau molculaire que pour le dveloppement de stratgies didentification ou de diagnostic. Avec la taille croissante des systmes dintrt, les techniques disponibles atteignent leur limite et la mise au point de mthodes alternatives devient ncessaire. Une dmarche efficace consiste combiner des techniques diffrentes donnant des informations complmentaires et orthogonales sur les molcules tudies. Cest dans cet esprit que nous avons dvelopp un nouveau dispositif exprimental qui permet de coupler un laser un spectromtre par mobilit ionique. La spectromtrie par mobilit ionique consiste sparer des ions selon leur vitesse de diffusion dans un gaz sous linfluence dun champ lectrique. A travers la mesure dune section efficace de collision avec le gaz, cette technique constitue une sonde globale de la conformation en phase gazeuse des ions tudis. Dans notre dispositif, le pouvoir de sparation de la mobilit ionique est utilis pour slectionner les molcules en conformation avant de provoquer leur fragmentation par absorption dun ou plusieurs photons. Lobjectif de ces expriences de fragmentation rsolue en conformation est dune part de comprendre linfluence de la conformation sur les canaux de fragmentation et dautre part dutiliser la sensibilit des sondes optiques larrangement local des molcules pour remonter une information structurale. La combinaison de sondes locale et globale de la conformation de biomolcules ouvre de nombreuses possibilits pour ltude ddifices molculaires complexes en permettant avec un mme instrument daccder diffrents niveaux de structuration. Nous prsenterons les premiers rsultats obtenus sur des sucres et des peptides modifis par greffage de chromophores. Ces systmes modles serviront de base lapplication de cette technique des protines entires et des complexes protine-ligand.
191/381
[P105] Outils statistiques pour la spectroscopie par mobilit ionique

Fabien Chirot1, Florent Calvo2, Florian Albrieux2, Yury Tsibyn3, Jrme Lemoine1, Philippe Dugourd2
1
2
Laboratoire de Spectroscopie Ionique et Molculaire, UMR 5579 CNRS-Universit Claude Bernard-Lyon1, 43 Boulevard du 11 Novembre 1918 , 69622 Villeurbanne, France
3
Biomolecular Mass Spectrometry Laboratory, Ecole polytechnique fdrale de Lausanne, EPFL SB ISIC LSMB BCH 4307 (Bt. BCH), 1015 Lausanne, Suisse Contact: Fabien Chirot (fabien.chirot@univ-lyon1.fr)
Keywords: Ionic mobility Linterprtation des expriences de mobilit ionique, et plus particulirement la dtermination de la structure des systmes tudis, requirent la mise en uvre doutils thoriques appropris. Une des principales difficults dans cette tche est de raliser une exploration efficace du paysage conformationnel des molcules dintrt, ce qui est dautant plus difficile que ces molcules sont complexes. Dans le cas des peptides ou des protines, une telle exploration nest ainsi ralisable, en pratique, que pour des entits de taille relativement modeste (une dizaine dacides amins). Parmi les outils thoriques disponibles, lutilisation de champs de forces dans le cadre dalgorithmes de dynamique molculaire par change de rpliques (REMD) est particulirement bien adapte pour traiter type de problmes. Nous montrerons que, au-del de son efficacit en termes dexploration conformationnelle, la REMD peut tre utilise pour obtenir des informations supplmentaires sur les systmes tudis. En effet, lutilisation de la mthode des historammes pondrs (WHAM) permet une exploitation plus fine des calculs REMD, en donnant accs des paramtres dordre autres que la section efficace de diffusion mesure en mobilit ionique. A travers lexemple de polyalanines (11 acides amins), nous illustrerons le fait que lobservation dun seul pic de mobilit ionique peut tre compatible avec la coexistence de diffrents conformres mis en vidence via une analyse par WHAM. Nous aborderons dautre part ltude de systmes plus complexes pour lesquels les simulations REMD ne parviennent pas reproduire des comportements tels que la bistabilit, pourtant observs exprimentalement. Dans ce cas, nous montrons lintrt dintroduire un biais dans le champ de force utilis afin de guider lexploration conformationnelle vers les zones correspondant aux observations exprimentales. Nous donnerons lexemple dun variant dune sous unit de la protine M2 du virus de la grippe A (25 acides amins) pour lequel les donnes exprimentales montrent la coexistence de deux structures alors que les simulations REMD nen rendent pas compte. La ralisation de simulations biaises permet dobtenir des conformations non-seulement compatibles avec lexprience, mais ayant de plus, pour certaines dentre elles, une nergie plus basse que celles ressortant de lexploration non biaise.
192/381
[P106] Development of a novel method for sterol analysis by UPLC-ESI-MS: Application on a neurodegeneration model
Sophie Ayciriex1, Mathieu Gaudin1, Fathia Djelti3, Mlaine Courcoul1, Anne Rgazetti1, Patrick Aubourg3, Nathalie Cartier3, Nicolas Auzeil1, Olivier Laprvote1
1
Laboratoire de Chimie-Toxicologie Analytique et Cellulaire EA4463, Universit Paris Descartes, Facult des Sciences Pharmaceutiques et Biologiques , 4, avenue de l'observatoire - case 47, 75006 Paris, France
2
Institut de chimie des substances naturelles, Centre de recherche de Gif, CNRS, 2, avenue de la terrasse, 91198 Gif-sur-Yvette cedex, france
3
INSERM UMR U745, Gntique et biothrapies des maladies dgnratives et prolifratives du systme nerveux, Universit Paris Descartes, 4, avenue de l'observatoire, 75006 Paris, France
4
Laboratoire de Toxicologie Biologique - Hpital Lariboisire, 2, rue Ambroise Par, 75975 Paris cedex 10, France Contact: Sophie Ayciriex (sophie.ayciriex@parisdescartes.fr)
Keywords: Separative analysis methods, High mass and high resolution mass Spectrometry, Organic and inorganic mass spectrometry Alzheimers disease (AD) is a progressive neurodegenerative disorder which conduces to dementia. The pathological hallmarks of AD include formation of extracellular neurotoxic amyloid plaques in the brain, dystrophic neurites associated with plaques and neurofibrillary tangles within nerve cell bodies. Cerebral cholesterol represents about 25% of the entire body cholesterol. It is mainly located in neuronal cell membranes and influences membrane stability and fluidity. Brain cholesterol is synthesized in situ. The excess of cholesterol is converted into 24S-hydroxycholesterol by neuronal cholesterol 24-hydroxylase (enzyme encoded by CYP46A1 gene) for elimination across the blood-brain barrier in the circulation. Some studies have revealed that disruption in cholesterol homeostasis could be involved in the pathogenesis of AD. Indeed, concentration modifications of cholesterol, 24S-hydroxycholesterol and other oxysterols, especially 7-ketocholesterol and 7--hydroxycholesterol are found in AD patients. These compounds seem to be key actors in this particular neurodegenerative disease. In order to quantify the cholesterol and his toxic metabolites in AD brain, we have developed a liquid chromatography method combined with high resolution mass spectrometry for the identification of oxysterols contained in brain of transgenic mouse models. The methodology includes a simple oxysterol derivatization step to add selectivity and enhance their ionization rates. This methodology was applied on a C57BL/6 mice model which underwent intra-cerebral injection of AAV5 vector encoding sh-CYP46A1 (RNA interference strategy) in hippocampus area. The effects of CYP46A1 gene down-regulation induce a neurodegeneration process due to the presence of toxic cholesterol metabolites.
193/381
[P107] Etude du mtabolome du slnium dans les levures par chromatographie liquide bimodale RPLC/HILIC couple la spectromtrie de masse haute rsolution (MSn Orbitrap)
Carine Arnaudguilhem1, Hugues Preud'homme1, Katarzyna Bierla1, Laurent Ouerdane1, Ryszard Lobinski1
1
Laboratoire de Chimie Analytique Bio-inorganique et Environnement IPREM UMR5254, 2 Avenue Pr. Angot, 64053 Pau, France Contact: Carine Arnaudguilhem (carine.arnaudguilhem@univ-pau.fr)
Keywords: Separative analysis methods, Organic and inorganic mass spectrometry Le slnium est un oligolment essentiel connu pour ses proprits anti-oxydantes, antitoxiques vis vis des mtaux, et son rle dans la prvention des cancers et des affections cardiovasculaires. Les levures enrichies en slnium constituent le complmentaire alimentaire le plus rpandu pour compenser les carences alimentaires, car elles sont capables d'accumuler jusqu' 3000mg Se/kg et sont peu onreuses. Le mtabolisme du slnium dpend fortement de sa forme chimique (spciation) et il tabli que seules les formes organiques sont assimilables. Ainsi, la dtermination du profil mtabolique du slnium, vritable empreinte de la levure et du processus de fermentation, est essentielle pour connatre la biodisponibilit du slnium. Cette tude a pour objectif de dvelopper une mthode d'analyse du mtabolome slni dans la fraction aqueuse de la levure. De nombreux travaux sont reports dans la littrature. Ils mettent gnralement en jeu des sparations par chromatographie en phase liquide en mode inverse, normal ou d'change d'ions. Ces mthodes ont permis d'identifier de nombreux mtabolites, mais elles sont spcifiques des molcules de mme proprits chimiques (apolaires, polaires ou ioniques). Dans cette tude, nous avons dvelopp une nouvelle mthode d'analyse par chromatographie liquide bimodale RPLC/HILIC couple la spectromtrie de masse haute rsolution MSn Orbitrap, permettant d'analyser simultanment les composs apolaires et polaires sur une mme colonne. Aprs fractionnement par chromatographie d'exclusion strique / ICP-MS, les mtabolites sont spars sur une colonne Hypercarb avec un gradient Eau 0,1% d'acide formique/actonitrile pour le mode RPLC et actonitrile/Eau 0,1% acide formique pour le mode HILIC. Cette colonne, constitue d'une phase inverse PGC (Porous Graphitic Carbon), prsente l'avantage de retenir galement les composs polaires et ioniques par des interactions lectrostatiques avec le carbone. Nous avons exploit cette proprit et utilis la colonne dans les 2 modes de fonctionnement, mode inverse et mode HILIC. Ce mode de sparation nous a permis de dtecter une quarantaine de composs slnis dont 7 en mode HILIC. Le couplage avec la spectromtrie de masse haute rsolution Orbitrap en mode MSn permet de caractriser et d'identifier les mtabolites avec une prcision de masse infrieure 2ppm. Link: www.lcabie.com
194/381
[P108] How proteomics shed light in understanding host-parasite interplay and clinical consequences during trypanosome infectious process.
Philippe Holzmuller1, Pascal Grbaut1, Nereida Parra Gimenez2, Jean-Paul Brizard3, Edith Demettre4, Martial Sveno4, Mary Ysabel Gonzatti2, Grard Cuny1
1 2
UMR InterTryp, CIRAD-IRD, Campus International de Baillarguet, 34398 Montpellier, France
Departamento de Biologa Celular, Universidad Simn Bolvar, Carretera Hoyo de la Puerta-Baruta, 1080 Caracas, Venezuela
3
Plate-forme 2D-DIGE, Ple Protomique de Montpellier (UMR5096), IRD, 911, avenue Agropolis, 34394 Montpellier, France
4
Plate-forme de Protomique Fonctionnelle (FPP), BioCampus Montpellier (UMS3426), 141, rue de la Cardonille, 34094 Montpellier, France Contact: Edith Demettre (edith.demettre@igf.cnrs.fr)
Keywords: Systems biology, Clinical proteomics, Bioinformatics et biostatistics dedicated to proteomics Animal trypanosomosis is one of the most severe constraints to agricultural development in Sub-Saharan Africa and is also an important disease of livestock in Latin America and Asia. The causative agents are various species of protozoan parasites belonging to the genus Trypanosoma, among which T. congolense and T. evansi are the major pathogenic species. The extracellular position of the trypanosomes in the hosts biological fluids implies to consider both the parasite and its excreted-secreted factors in the infectious process and its physiopathological consequences. Despite decades of research, the molecular keys inducing clinical diversity are still poorly understood. The postgenomic era have stimulated the development of new techniques and bioinformatics tools to identify the locations, functions and interactions of the gene products in tissues and/or cells of living organisms. Among these advances, we used two-dimensional Differential In Gel Electrophoresis (2-DIGE) coupled to Mass Spectrometry (MS), a well-proven technology to propose for the first time a comparative approach of the secretome (i.e. naturally excreted-secreted molecules) of T. congolense and T. evansi clones exhibiting marked differences in their virulence and pathogenicity profiles. Surprisingly, the 2D-DIGE/MS analytical filter highlighted few differentially expressed molecules, some of which were moreover identified as Putative Uncharacterised Protein. Nevertheless and interestingly, bioinformatics allowed us to directly link several proteins to the clinical disorders observed in trypanosome-infected animals in the field. As illustrated by four biomarkers, it shows how proteomics is powerful in the molecular identification of differentially expressed trypanosomes molecules correlated with either the virulence process or exhibiting potential properties to induce pathogenic dysregulation of physiological functions. Moreover, deciphering of the molecular dialogues and conflicts that govern host-parasite interplay is promising to define new molecular targets for improved field diagnosis and new strategies of interference with the infectious process to fight against animal trypanosomosis.
195/381
[P109] Troglitazone metabolism in a chimeric mouse model with humanized livers determined by high mass accuracy MSn analysis
Alan BARNES1, Neil LOFTUS1, Chris TITMAN1, Ian D. WILSON2, Filippos MICHOPOULOS2, Timothy SCHULZUTERMOEHL2, Yoshio MORIKAWA3, Stphane MOREAU4
1 2 3 4
SHIMADZU ISS, Trafford Wharf Road, M17 1GP MANCHESTER (UK) ASTRA ZENECA, Alderley Park, Macclesfield , SK10 4TG Cheshire (UK) PhoenixBio Co. Ltd, 3-4-1, Kagamiyama, 739-0046 Higashi-Hiroshima City (Japan) SHIMADZU France, 65 Avenue du General De Gaulle, 77420 CHAMPS/MARNE (France)
Contact: Stphane MOREAU (sm@shimadzu.fr)
Keywords: High mass and high resolution mass Spectrometry Introduction The chimeric PXB mouse model has a high percentage of the liver repopulated by human hepatocytes and has been proposed as a tool for the prediction of human disposition and hepatic effects. To assess the PXB model, a known human selective hepatotoxin, troglitazone, was dosed to wild type (SCID) and PXB mice (n=3 or 4 per group and dose level). This paper describes the metabolite changes between SCID and PXB mice following a single daily oral administration for 7 days of either vehicle or 300 or 600mg/kg/day of troglitazone using high mass accuracy MSn analysis. Method Liver extracts from SCID and PXB mice were analysed using a high resolution LC/MSn system (Nexera LC coupled with a LCMS-IT-TOF; Shimadzu Corporation). Both aqueous and organic extracts were analysed using a Phenomenex Kinetex C18 1.7um (2.1x100mm) held at 60C; components were separated with a gradient elution (mobile phase was A - water + 0.1% formic acid and B - methanol:propan-2-ol (85:15) + 0.1% formic acid) at a flow rate of 0.6ml/min. The LCMS-IT-TOF acquired MS and MS2 data in both positive and negative ionisation using a polarity switching speed of 100msecs with a scan range of m/z 150-1250. Profiling Solution software (Shimadzu, Japan) was applied to metabolite profiling analysis to assess the impact of changes in lipid profiles. Results Troglitazone (CS-045) is an oral antidiabetic agent with hypoglycemic activity in non-insulindependent diabetes mellitus in animals and humans. The pharmacodynamics of troglitazone in man are characterized by metabolism in the liver, CLtot essentially reflects hepatic metabolism. Glucuronidation and sulfation are the major pathways of metabolism, along with a minor degree of oxidation to the quinone form. SCID wild type mice showed liver metabolite profiles which are consistent with previously published data but differ from human metabolite profiles. PXB mice metabolite profiles differ markedly from the wild type. Although sulfate metabolite dominates the metabolism pathway in PXB mice, the glucuronide and sulfate metabolites are minor metabolites in comparison to the SCID wild type mouse. A finding which is consistent with published data in man. In conclusion, the PXB mouse model can provide a first insight into human in vivo metabolism. Link: www.shimadzu.com
196/381
[P110] Non Card Dried Biofluid Spot Format for the Analysis of Protease Inhibitors using High resolution Liquid chromatography with Fast Tandem Mass Spectrometric Detection.
Michel WAGNER1, Neil LOFTUS2, Alan BARNES2, Chris TITMAN2, Emmanuel VARESIO1, Grard HOPFGARTNER1, Stphane MOREAU3
1 2 3
UNIVERSITY OF GENEVA, Quai Ernest-Ansermet 30, CH-1211 GENEVA 4 (Suisse) SHIMADZU , Mill Court, Featherstone Road, Wolverton Mill South,, MK12 5RD MILTON KEYNES (UK) SHIMADZU France, 65 Avenue du Gnral De Gaulle, 77420 CHAMPS/MARNE (France)
Contact: Stphane MOREAU (sm@shimadzu.fr) Keywords: Proteins, peptides and small molecules quantification Introduction The use of dried biofluid spot (DBS) on filter paper is rapidly becoming the method of choice for the analysis of pharmaceuticals and their metabolites by HPLC coupled to MS detection. The assay typically uses a punched disk taken from the DBS sample, followed by solvent-liquid extraction and analysis by reversed phase HPLC separation, combined with multiple reaction monitoring mass spectrometric detection. Unfortunately, SLE is not very selective and can lead to possible ionization matrix effects. We present a novel, tube based format for DBS, which allows sample collection on paper and analyte extraction, in a single device. This eliminates the need of the punching step and allows novel approaches in sample storage and analyte extraction. In this paper we describe sample preparation strategies followed by fast chromatographic separation on a triple quadrupole instrument in the SRM mode with very short duty cycle and fast polarity switching. Methods DBS extracts were analyzed on a Nexera UHPLC system equipped with a fast injector (SIL30AC-MP) hyphenated to a LCMS-8030 triple quadrupole mass spectrometer (Shimadzu). Mobile phases were: A) 0.1% acetic acid (AA) in water and, B) 0.1% AA in acetonitrile. The flowrate was set at 600 l/min. The binary gradient was linear from 30% to 80% in 3 min. for protease inhibitors assays and from 30% to 70% B in 2.5 min. for bosentan assay. In both cases the column was a Kinetex XB-C18 (2.1 x 100 mm, 1.7 m, Phenomenex) and was thermostated at 50C. The injection was of 5 l. The total MS duty cycle was of 184ms including eight 20ms SRM transitions for the PI assay (electrospray ionisation in positive polarity). For the bosentan assay the total MS duty cycle was of 318ms for eight 20ms and eight 10ms SRM transitions with electrospray alternating from positive to negative polarity. Results A key advantage of this technique is that DBS tube-based format allows sample collection on paper and analyte extraction in a single device. Using typical sample volumes of 15 l allows LOQ down to the low ng/ml range to be achieved. Variable filter thicknesses can be used from 5-25 l to meet the needs of the individual assay. This approach can be used not only to whole blood but to all other biofluids using a generic SLE sample preparation method which could be a considerable advantage in clinical analysis. To achieve short analysis time and high separation efficiencies, 2 mm i.d. core-shell silica based columns where used at a flow rate of 0.6 mL/min and pressures of about 600 bars at a temperature of 50 C (shorter analysis time could be achieved by even increasing further the flow rate). Optimizing response of the MS used a high speed data acquisition and fast polarity switching (15msecs). Using a 15L blood or plasma aliquot adequate sensitivity, precision and accuracy could be achieved for the determination of seven protease inhibitors using only one internal standard.
197/381
[P111] tude de complexes biologiques non-covalentS de hauts poids molculaires par MALDI-TOF-MS aprs pontage chimique.
Reynald Hlye1, Mlanie Lebreton 1, Matthieux Bruneaux2, Franck Zal2, Noelle Potier1, Emmanuelle Leize1
1 2
LDSM2, Institut de Chimie, UDS/CNRS UMR 7177, 1 rue Blaise Pascal, 67000 Strasbourg, France quipe d'cophysiologie, UPMC/CNRS 7144, Station Biologique, 29682 Roscoff, France
Contact: Reynald Hlye (reynald.helye@hotmail.fr)
Keywords: High mass and high resolution mass Spectrometry, Systems biology Depuis quelques annes, la spectromtrie de masse (MS) sest impose comme une alternative fiable pour ltude des complexes non covalents et des mthodologies ont t dveloppes pour dmontrer lexistence dun complexe ou en dterminer la stchiomtrie. Les possibilits de la MS ont rgulirement progress au fil des annes, sattaquant des complexes toujours plus lourds ou labiles. Toutefois, la technique se heurte certaines difficults. En effet, la grande majorit des tudes biologiques sont effectues par ionisation lectrospray (ESI-MS). Nanmoins, cette source est trs sensible la prsence de sels non volatils ce qui ncessite des tapes de dessalage souvent longues et parfois critiques pour la stabilit du complexe en solution. Dautre part, ltude de complexes transitoires ou de dure de vie courte ne peut tre aborde par cette approche. Cest pourquoi nous nous sommes intresss une stratgie permettant de stabiliser le complexe de faon irrversible : le pontage chimique (ou cross-linking ). Une fois le pontage ralis entre les diffrents partenaires, nous pouvons nous affranchir de ltape de dialyse dans un tampon de sels dammonium et prparer lchantillon avec davantage de libert pour en dterminer la stchiomtrie dinteraction par MALDI-MS. Dans cette optique, deux kits de pontage fournis par la socit CovalX (K100 et K200 ; Zurich, Suisse) ont t tests. Les conditions exprimentales de pontage chimique ont t optimises sur des complexes de stchiomtries connues : le rcepteur de lacide rtinoque (RAR) et lalcool dshydrognase de levure (yADH). Linfluence de plusieurs paramtres sur le rendement de pontage mais galement sur la spcificit dinteraction a t value : le ratio entre lagent pontant et le complexe, sa ractivit, la temprature et le temps de raction notamment. Cette stratgie a ensuite t applique ltude de complexes plus lourds, tels que les hmocyanines du crabe Carcinus maenas (~900 kDa). Les rsultats obtenus sont encourageants mme si des expriences strictes de contrle doivent tre effectues afin de vrifier que les spectres de masse obtenus refltent les abondances des espces prsentes en solution.
198/381
[P112] tude par analyse protomique de biomarqueurs dexposition des cancrignes.

Marianne Ibrahim1, Lauriane Kuhn2, Philippe Hammann2, Zeina Dagher3, Emmanuelle Leize1
1 2 3
LDSM2, Institut de Chimie, UDS/CNRS UMR 7177, 1 rue Blaise Pascal, 67000 Strasbourg, France Plateforme Protomique (IBMC), 15 rue R. Descartes, 67000 Strasbourg, France Dpartement de Biologie, Facult des Sciences, Universit Libanaise, B.P. 90656 Fanar, Liban
Contact: Emmanuelle Leize (npotier@chimie.u-strasbg.fr)
Keywords: Organic and inorganic mass spectrometry, Systems biology Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants ubiquitaires de lenvironnement montrant une forte toxicit ; certains dentre eux sont suspects dtre cancrignes pour lhomme. Dans cette tude, nous avons effectu des analyses protomiques sur un modle de cellules hpatiques (HepG2) exposes au Benzo(a)pyrne (BaP), un cancrigne pour lHomme (class groupe 1 selon le Centre International de la Recherche sur le Cancer). Notre approche in vitro vise identifier et quantifier, par spectromtrie de masse, de nouveaux biomarqueurs dans ces cellules exposes au BaP 1M. Le test de viabilit cellulaire na montr aucun effet cytotoxique du BaP cette concentration. Les analyses protomiques utilisant llectrophorse bidimensionnelle ont montr des spots dintrt qui ont t analyss par nano-LC/MSMS. Nous avons identifi deux protines pouvant tre utilises comme biomarqueurs potentiels prcoces applicables dans la recherche sur le cancer : RuvB-like2 et Translationally Controlled Tumor Protein (TCTP). En perspectives, nous souhaitons valider ces biomarqueurs dans les milieux biologiques des populations exposes au HAP, en environnement gnral et/ou professionnel.
199/381
[P113] Spectromtrie de masse MALDI de polymres synthtiques fonctionnaliss par un nitroxyde

Caroline Barrre1, Christophe Chendo2, Valrie Monnier2, Thomas Trimaille3, Trang N.T. Phan3, Didier Gigmes3, Stphane Viel1, Stphane Humbel4, Denis Bertin3, Laurence Charles1
1
Laboratoire Chimie Provence, UMR 6264, Equipe Spectromtries Appliques la Chimie Structurale, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille
2
Spectropole, Fdration des Sciences Chimiques-CNRS, FR 1739, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille
3
Laboratoire Chimie Provence, UMR 6264, Equipe Chimie Radicalaire, Organique et Polymres de Spcialit, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille
4
Institut des Sciences Molculaires de Marseille, UMR 6263, Equipe Chimie Thorique et Mcanismes, Campus Scientifique de St Jrme - Av. Escadrille Normandie Niemen, 13397 Cedex 20 Marseille Contact: Caroline Barrre (caroline.barrere@etu.univ-provence.fr)
Keywords: Organic and inorganic mass spectrometry La polymrisation radicalaire assiste par nitroxyde (NMP) permet la prparation de copolymres synthtiques de taille contrle. Dans cette tude, la synthse dun copolymre bloc poly(oxyde dthylne)/polystyrne (POE-b-PS) est ralise en faisant crotre le bloc PS partir dun homopolymre POE fonctionnalis avec le nitroxyde SG1 (N-(2-mthylpropyl)-N-(1dithylphosphono-2,2-dimthyl propyl)-N-oxyl). Le contrle analytique de la fonctionalisation du POE est donc fondamental et consiste typiquement dterminer la masse des groupements terminaux partir dun spectre de masse. Cependant, la fragilit de la liaison C-ON qui lie le groupement SG1 au squelette polymrique, et sur laquelle repose le processus NMP, devient un problme majeur pour lanalyse MALDI car le nitroxyde est systmatiquement limin sous forme radicalaire lors du processus dionisation. Pour produire des adduits oligomriques intacts en phase gazeuse et ainsi assurer la fiabilit de lanalyse des groupements terminaux, les facteurs influant sur la stabilit de la liaison C-ON ont t tudis. Une tude ab initio a montr une augmentation de lnergie de dissociation de cette liaison lorsque le groupement tert-butyl port par lazote du nitroxide est substitu par un atome dhydrogne. Un traitement lacide trifluoroactique a donc t dvelopp pour oprer cette substitution, transformant efficacement le groupement SG1 en un groupement SG1. Les spectres MALDI-MS obtenus pour les homopolymres POE-SG1 indiquent que le renforcement de la liaison C-ON prdit par les calculs thoriques est suffisant pour permettre la dsorption dadduits intacts du polymre trait.
200/381
[P114] Dveloppements mthodologiques en MS/MS pour la production de matriaux bio-ressourcs

Mohamed Major1, Guillaume Moreira2, Catherine Lefay2, Denis Bertin2, Didier Gigmes2, Laurence Charles1
1
Laboratoire Chimie Provence, UMR 6264, Equipe Spectromtries Appliques la Chimie Structurale , AixMarseille Universit I, II et III CNRS, Campus de St Jrme, 13397 Marseille
2
Laboratoire Chimie Provence, UMR 6264, Equipe Chimie Radicalaire-Organique et Polymres, Aix-Marseille Universit I, II et III CNRS, Campus de St Jrme, 13397 Marseille Contact: Mohamed Major (mohamed.major@etu.univ-provence.fr)
Keywords: Organic and inorganic mass spectrometry Lengouement actuel pour la production de polymres partir de ressources renouvelables, telles que la cellulose, vise la fois minimiser notre dpendance vis--vis des drivs ptroliers et rpondre des enjeux environnementaux. Puisque les proprits mdiocres des polymres naturels limitent fortement leurs applications, une stratgie prometteuse consiste laborer des matriaux rsultant de mlanges cellulose/polymres synthtiques. Cest un moyen simple de diminuer la proportion de drivs ptroliers dans des matriaux dusage courant tout en amliorant leur bio-dgradabilit. Afin dassurer la stabilit de ces mlanges, il est ncessaire dajouter des composs amphiphiles, miscibles dans chacun des constituants. Pour ce faire, de nombreux travaux explorent la possibilit de modifier la cellulose mais se heurtent lextrme complexit structurale et la faible solubilit de ce polymre naturel. Une alternative intressante consiste produire des macromolcules hybrides, constitues de chanes polysaccharides plus courtes et greffes par des segments synthtiques. Le potentiel stabilisant de ces co-polymres tant intimement li leurs caractristiques structurales (taille du polysaccharide, longueur et nombre de greffons synthtiques, ), la mise au point des stratgies de synthse doit se faire de concert avec le dveloppement des mthodes analytiques. Dans ce cadre, la spectromtrie de masse savre une technique de choix, aussi bien pour caractriser la masse des molcules en mode MS que pour localiser les greffons en mode MS/MS. Cette tude prsente les rsultats prliminaires obtenus pour des sucres de faible taille, glucose et cellobiose, fonctionnaliss avec des groupements acrylate. En effet, la synthse des copolymres greffs polysaccharides/polystyrne est base sur la polymrisation radicalaire contrle assiste par nitroxide, pour permettre la croissance du polystyrne basse temprature, une condition cruciale au regard de la stabilit thermique des polymres naturels. Une srie de molcules comportant un nombre croissant de groupements acrylate greffs sur une mme unit glucose ont t synthtises puis examines par spectromtrie de masse. Loptimisation des conditions exprimentales est discute, notamment le mode de polarit des analyses, et les voies de fragmentation de ces molcules sont tudies en fonction de leur degr de substitution, dans des conditions de dissociation induite par collision.
201/381
[P115] Structuration des dpts MALDI : une tude multidisciplinaire en phase solide
Yannis Major1, Hlne Pizzala1, Fabio Ziarelli2, Christian Dominici3, Trang Phan4, Laurence Charles1
1
Laboratoire Chimie Provence, UMR 6264, Equipe Spectromtries Appliques la Chimie Structurale, AixMarseille Universit I, II et III CNRS, Campus de St Jrme., 13397 Marseille, France
2
Fdration des Sciences Chimiques CNRS, FR1739, Spectropole, Aix-Marseille Universit I, II et III CNRS, Campus de St Jrme., 13397 Marseille, France
3
Fdration des Sciences Chimiques CNRS, FR1739, Centre Pluridisciplinaire de Microscopie Electronique et de Microanalyse, Aix-Marseille Universit I, II et III CNRS, Campus de St Jrme., 13397 Marseille, France
4
Laboratoire Chimie Provence, UMR 6264, Equipe Chimie Radicalaire, Organique et Polymres de Spcialit, Aix-Marseille Universit I, II et III CNRS, Campus de St Jrme., 13397 Marseille, France Contact: Yannis Major (yannis.major@etu.univ-provence.fr)
Keywords: Organic and inorganic mass spectrometry La technique MALDI s'est rapidement impose comme la mthode de choix pour l'ionisation des macromolcules dorigine biologique ou synthtique. Elle se prte particulirement bien lionisation dchantillons complexes tels que les polymres synthtiques car elle permet la production dadduits monochargs partir de chaque oligomre, donnant lieu des spectres de masse plus simples que ceux obtenus par electrospray. Toutefois, la mauvaise connaissance des processus fondamentaux qui rgissent la dsorption et l'ionisation des analytes devient un problme majeur qui limite lapplication gnralise de la technique MALDI aux polymres synthtiques. Le point-cl de la russite d'une analyse MALDI-MS rside dans la prparation des chantillons. La matrice et lagent de cationisation, ainsi que leur concentration relative, doivent tre choisis en fonction de la nature et de la taille du polymre mais cette optimisation reste extrmement empirique car limpact de chacun de ces paramtres reste mconnu. De plus, le mode de prparation des chantillons MALDI influence galement le rendement de lionisation et la nature des ions forms. Cette tude propose dexaminer des chantillons MALDI, tant au point de vue morphologique que microstructural, avec des techniques ddies lanalyse des solides afin dtablir des relations entre la structure des dpts MALDI, leur mode de prparation et lefficacit du processus dionisation. Afin de limiter les facteurs associs au mode de prparation, notamment ceux lis lutilisation dun solvant, les chantillons tudis sont obtenus par broyage au vortex (mthode "solvent-free"). La composition des chantillons a t choisie pour que chaque technique mise en uvre puisse apporter des informations complmentaires : ils sont constitus de 2,5-DHB (matrice), de CsCl (agent de cationisation) et de poly(oxyde dthylne) (analyte). La morphologie, l'organisation et l'homognit de l'chantillon ont t explores par microscopie lectronique balayage (MEB). La calorimtrie diffrentielle balayage (DSC) a t utilise pour mettre en vidence la mobilit des chanes de polymre. Enfin, les interactions intermolculaires et la mobilit des diffrentes espces ont t sondes par RMN du solide (13C et 133Cs).
202/381
[P116] Analyse comparative du sous-protome cytoplasmique de la Barrire Hmato-Encphalique: approche label free et approche par marquage isotopique stable.
Barbara Deracinois1, Sophie Duban-Deweer 1, Johan Hachani1, Romo Cecchelli1, Christophe Flahaut1, Yannis Karamanos1
1 2 3
Universit Lille Nord de France, 1bis rue Georges Lefvre, 59044 Lille Universit dArtois, LBHE, Rue jean Souvraz, 62307 Lens IMPRT-IFR 114, CHRU de Lille, 59000 Lille
Contact: Christophe Flahaut (christophe.flahaut@univ-artois.fr)
Keywords: Systems biology, Proteins, peptides and small molecules quantification 95 % des composs thrapeutiques vise crbrale sont inefficaces in vivo du fait de la prsence de la Barrire Hmato-Encphalique (BHE) quils sont incapables de franchir. Bien que relativement connu dans son aspect physiologique, le phnotype BHE des cellules endothliales des capillaires crbraux (BCECs) reste mal compris au regard des mcanismes molculaires qui gouvernent son tablissement et son maintien. Dans cette optique, laide de modles diffrentiels de BHE in vitro (solo-culture de BCECs bovines ou co-culture avec des cellules gliales de rat), nous avons initi deux tudes protomiques comparatives afin didentifier les protines cytoplasmiques potentiellement impliques dans ltablissement et le maintien de ce phnotype. Les protines, extraites au Triton X-100, sont soumises un fractionnement organique avant analyses par chromatographie liquide couple la spectromtrie de masse (1D-LC MALDIMS/MS). Lapproche sans marquage, dite label free , consiste en une simple analyse comparative des protines identifies. La seconde approche de type Isotope Coded Protein Label (ICPL), consiste quantifier les protines grce un marquage isotopique effectu au pralable. Lefficacit et la reproductibilit du fractionnement protique utilis sont dmontres par SDSPAGE, 2D-PAGE et via lanalyse compare des donnes de masse obtenues partir de 3 sries exprimentales. Lapproche label free des cellules ayant perdu certaines proprits de barrire (solo-culture de BCECs) ou non (co-culture) a permis lidentification de 440 et 410 protines respectivement. Comparativement, 11 protines semblent plus abondamment retrouves au sein des BCECs co-cultives, contre 41 protines dans les BCECs solo-cultives. Lapproche ICPL est quant elle complmentaire et indique que 15 protines sont potentiellement sur-exprimes et 43 protines sous-exprimes dans les BCECs co-cultives. Les variations quantitatives de deux protines dun intrt particulier dans le domaine de la BHE (la phosphatase alcaline et lADP-ribosylation factor 4) ont t values par PCR et western-blot.
203/381
[P117] Virus-Like Particles in Parasitoids' Venom: Viral or Cellular Origin?

Emilie Goguet-Surmenian1, Maya Belghazi2, Marc Ravallec3, Christian Rebuf1, Dominique Colinet1, Marylne Poiri1, Jean-Luc Gatti1
1 2
ESIM, UMR 1301 INRA-CNRS-Universit Nice-Sophia Antipolis, 400 route des chappes, 06903 sophia antipolis
Centre d'Analyses Protomiques de Marseille (CAPM) IFR Jean Roche Facult de Mdecine - Secteur Nord Universit de la Mditerrane , CS80011 Bd Pierre Dramard , 13344 MARSEILLE Cedex 15
3
UMR 1333 Diversit, INRA-Universit Montpellier 2, Place Eugne Bataillon, 34 095 Montpellier Cedex 5
Contact: Jean-Luc Gatti (Jean-luc.gatti@sophia.inra.fr)
Keywords: Systems biology During egg oviposition, endoparasitoid wasps inject ovarian and venom compounds that will regulate the host physiology and protect the egg from the host immune response. For instance, Leptopilina boulardi strains, parasitoids of Drosophila, inject venom that contains specific toxic proteins and vesicles devoid of DNA or RNA named Virus-Like Particles (VLPs). Some of these VLPs have been demonstrated to participate in suppressing the host immune defense, likely by convoying proteins that destroy or inactivate the cellular host response. Under electron microscopy, we observed that these purified VLP are heterogeneous in form and size between different L. boulardi strains. We have analyzed by a proteomic approach the composition of the VLPs-containing pellet obtained by centrifugation of the content of venom reservoir of L. boulardi strains ISm and ISy. We demonstrate that the major immunosuppressive factor of L. boulardi Ism, a RacGAP protein named LbGAP, is mainly associated with the VLP pellet. None of the major proteins identified in VLPs matched with known viral proteins, which strongly suggests that these vesicles are cellular and not viral products. Then we suggest the name of venosomes for the vesicles observed in the venom of these species to avoid confusion with true viral particles found in other parasitoid species. These venosomes may serve as a transport and targeting system for specific venom components that are necessary for parasitism success in this species.
204/381
[P118] Influence des interactions de type pont salin lors de la fragmentation de complexes peptides-ADN en phase gazeuse
Bessem BRAHIM1, Sandra ALVES1, Jean-Claude TABET1
1
Laboratoire de Chimie Structurale Organique et Biologique, 4 place Jussieu, 75252 Cedex 05 Paris, FRANCE
Contact: Bessem BRAHIM (bessem.brahim@upmc.fr)
Keywords: Systems biology Tous les mcanismes cellulaires des organismes vivants sont issus dinteractions non-covalentes (NCI) au niveau molculaire que ce soit, entre une protine et un double brin dADN (rgulation de lexpression des gnes), entre une protine et un simple brin dARN (tape de traduction) ou encore entre diffrentes protines. Dans cette tude, lorigine des interactions existantes entre protine et ADN a t examine en phase gazeuse par ESI sous mode positif et ngatif, afin de prserver les interactions ioniques entre les rsidus basiques (R/K) et les groupements phosphates de lADN et de les mettre en vidence. Cela permettra de dmontrer lexistence de formes zwitterioniques en phase gazeuse, notion encore dbattue dans le cadre de lADN. Diffrentes combinaisons (i) de peptides de longueurs et de basicits variables et (ii) doligonuclotides de longueurs et daffinits protoniques variables, ont t testes. Le transfert en phase gazeuses a t ralis en mode positif et ngatif grce une source electrospray combine spectromtre de masse LTQ-Orbitrap permettant la mise en uvre dexpriences de MSn dans le pige linaire (LTQ) par excitation rsonante radiale (CID) et/ou par dissociations induites par transfert dlectron (ETD). Les spectres CID des complexes peptides/ADN (simple et double brins) ont t enregistrs haute rsolution grce lOrbitrap (FT-MS) afin de faciliter linterprtation. De prcdentes tudes ont montr que la fragmentation des complexes multi-dprotons est strictement dpendante des proprits basiques des rsidus et des proprits acides des nuclotides, suggrant la prsence de fortes interactions de types pont salin entre les brins dADN et les peptides. Des ions fragments abondants conservant ces types dNCI, provenant de ruptures de liaisons peptidiques et/ou nuclotidiques via libration de nuclobase, ont t dtects et dmontrent la force de ces NCI. La comparaison du comportement des ions, dans les modes dionisation positive et ngative, a montr des voies de fragmentation similaires du complexe, cohrentes avec la prservation des NCI prsentes en solution. Le peptide semble donc tre fix une position de lADN dicte par sa conformation en solution. De plus, du fait des processus de dsolvatation, les interactions lectrostatiques renforces en phase gazeuse permettent de bloquer la position du peptide sur lADN, ce qui explique que des liaisons covalentes soient rompues sous activation en MS/MS. Il a aussi t not que lexcs de charges (positive ou ngative) et la prsence dNCI orientent les voies de fragmentations vers des ruptures multiples de liaisons nuclotidiques, fournissant un nombre considrable dinformations conformationnelles. En conclusion, cette tude montre que lESI est une mthode suffisamment douce pour conserver les NCI mais aussi une grande partie des conformations prexistantes en solution.
205/381
[P119] Composition membranaire et internalisation des peptides vecteurs : Quantification par spectromtrie de masse MALDI-TOF
Chrine Bechara1, Yefim Zaltsman1, Sandrine Sagan1
1
Laboratoire des BioMolcules, 4 Place Jussieu, 75005 Paris
Contact: Chrine Bechara (cherine.bechara@upmc.fr)
Keywords: Proteins, peptides and small molecules quantification Les peptides vecteurs sont des petites squences dacides amins basiques et plus ou moins amphiphiles. Ces peptides sont capables de transporter des molcules biologiquement actives, gnralement conjugues de manire covalente lintrieur des cellules selon deux grandes voies dinternalisation, lendocytose et la translocation directe. La balance entre ces deux voies (endocytose et translocation directe) dpend de plusieurs paramtres, notamment de la squence du peptide vecteur, de la nature de la cargaison conjugue, du type cellulaire, de la nature de la liaison entre le peptide vecteur et la molcule conjugue, de telle sorte que la localisation finale intracellulaire de la molcule cargaison ne peut tre prdite. Sur cette base, il est difficile de rationaliser un adressage spcifique vers une activit biologique intracellulaire cible avec cette stratgie covalente. Dans ce contexte, nous nous intressons aux paramtres qui influent sur les voies dinternalisation et donc la localisation intracellulaire des peptides vecteurs et/ou de leurs conjugus. Les glycoconjugus en surface cellulaire sont les premires molcules interagissant avec un peptide vecteur. Ces carbohydrates pourront tre des effecteurs dendocytose ou important pour la translocation en concentrant localement le peptide afin quil puisse interagir avec les lipides membranaires en vue dinternalisation. Nous sommes donc intresss par ltude de leffet de diffrents carbohydrates sur les voies dentre. Linternalisation est suivie par une quantification directe du peptide intracellulaire par spectromtrie de masse. De plus, laffinit de ces peptides aux diffrents polysaccharides a t mesure. Les rsultats obtenus feront lobjet de cette prsentation.
206/381
[P120] Label free quantification of coat proteins of Bacillus cereus affected by sporulation temperature
Stella Planchon1, Cline Henry2, Benot Valot2, Isabelle Bornard3, Jean-Franois Maingonnat1, Frdric Carlin1, Vronique Broussolle1
1
INRA, UMR 408 Scurit et qualit des produits d'origine vgtale, Universit d'Avignon et des Pays de Vaucluse, 84000 Avignon
2
INRA, Plateforme d'Analyse Protomique Paris Sud Ouest, UMR 1319 MICALIS, Domaine de Vilvert, 78352 Jouy en Josas
3
INRA, Unit de Pathologie Vgtale, Plateau de microscopie, , 84143 Montfavet
Contact: Cline Henry (celine.henry@jouy.inra.fr)
Keywords: Proteins, peptides and small molecules quantification, High mass and high resolution mass Spectrometry Sporulation temperature affects significantly B. cereus KBAB4 spore properties such as germination to alanine and resistance to heat, to pulsed light and to chemical treatments (Planchon 2011). Physiological differences could be due to ultra structural differences between spores produced at two sporulation temperatures. Observations of spore cross-sections in transmission electron microscopy showed a thicker outer coat layer in spores produced at low temperature, suggesting variations in coat protein composition. Coat protein-containing fractions of B. cereus KBAB4 spores produced at 10C and 30C were analysed by 1D gel. Bands were excised, digested and subjected to LC-MS/MS analysis using the U3000 nano-HPLC system (Dionex, Amsterdam, Netherlands) and the LTQ-Orbitrap (Thermo Scientific, Bremmen, Germany). Subsequently, 1D-gel coupled with identification and label-free quantitation with LTQ-Orbitrap was carried out using spectral counting an integration of peak area. The originality of this label-free quantitation is that we used X!tandem software for identification of proteins coupled with a module of peptide retention time alignment and integration of peak area adapted by the PAPPSO platform : MassChroQ*. Among the 200 identified proteins, 64 proteins varied with the sporulation temperature, including 13 coat proteins, with CotS and CotSA only recovered in spores produced at low temperature. The CotE and CotG proteins are present in higher quantity in spores produced at 10C and could be involved in the ultrastructural modifications of the outer coat layers. Moreover, 7 proteins are found in lower quantity in spores produced at 10C, 4 have unknown role in coats while Cot, CwlJ and SleL are clearly involved either in spore resistance and germination of B. subtilis and B. anthracis. Further investigations are needed to understand the role of the coat proteins affected by the sporulation temperature in B. cereus resistance. Planchon S. et al. (2011) Spores of Bacillus cereus strain KBAB4 produced at 10C and 30C display variations in their properties. Food Microbiology 28(2):291-297. *http://pappso.inra.fr/bioinfo/
207/381
[P121] Simulations de dynamique directe pour tudier la dissociation induite par collisions des biomolcules
Jean-Yves Salpin1, Yannick Jeanvoine1, Marie-Pierre Gaigeot1, Riccardo Spezia1
1
Laboratoire Analyse et Modlisation pour la Biologie et l'Environnement - LAMBE - UMR 8587 - Universit d'Evry Val d'Essonne, Boulevard Franois Mitterrand, 91025 Evry Contact: Jean-Yves Salpin (jean-yves.salpin@univ-evry.fr)
Keywords: Instrumentation, Organic and inorganic mass spectrometry La spectromtrie de masse est devenue un outil danalyse incontournable. Outre son intrt sur le plan purement analytique (caractrisation et quantification des composs), cette technique permet galement daborder ltude de la ractivit en phase gazeuse despces ionises allant de quelques atomes des systmes complexes (biopolymres, polymres de synthse, agrgats organiques et inorganiques). La modlisation molculaire est un outil indispensable pour linterprtation au niveau molculaire de ces processus. Les mthodes de la chimie quantique sont utilises de faon courante pour les interprter. Il est cependant indispensable, pour une meilleure interprtation des rsultats observs, de considrer aussi laspect dynamique. Dans ce but, nous avons utilis une approche de dynamique directe qui traite de faon explicite la collision entre lion et le projectile de gaz rare au cours de laquelle une certaine quantit d'nergie est transfre lion, conduisant ainsi sa fragmentation. Grce aux tudes de dynamique directe de la fragmentation, il est possible davoir accs diffrents dtails au niveau microscopique sur les ions soumis au processus CID, tels que la structure des fragments obtenus, la section efficace du CID, les mcanismes de fragmentation ou encore la diffrence de ractivit des isomres. Le transfert dnergie et la fragmentation de diffrents systmes (ure protone, complexe [Ca(ure)]2+ ou encore des peptides modles protons) ont t tudis. Nous avons dans ce but utilis une approche QM/MM o linteraction projectile/ion est trait par une potentielle analytique, et lion par une mthode quantique, au niveau MP2 pour les petits systmes et smiempiriques (AM1 et PM3) pour les grandes systmes. Les simulations ont mis en vidence que deux mcanismes de fragmentation sont possibles: (i) le mcanisme dit shattering, par lequel les produits sont obtenus juste aprs une priode vibrationnelle du "stretching" de la liaison qui va se casser, et (ii) un mcanisme de transfert dnergie (qui nest pas un IVR complet) qui donne les produits aprs plus dune priode de vibration. Les ions fragments observs exprimentalement et correspondant aux processus de plus haute nergie, sont obtenus grce au mcanisme de shattering. Ceci est un rsultat important car il suggre que les approches cintiques bases sur la limite statistique de la raction unimolculaire (RRKM par exemple) peuvent ne pas tre correctes pour dcrire la fragmentation unimolculaire.
208/381
[P122] Pipeline applicatif pour laide a lanalyse de donnees de spectrometrie de masse en proteomique clinique
Valentin Bouquet1, Pierre Naubourg2, Caroline Truntzer3, Isabelle Vergely1
1 2
ASA - Advanced Solutions Accelerator, 199 rue de l'Oppidum, 34170 Castelnau le Lez, FRANCE
LE2I - Laboratoire Electronique, Informatique et Image - UMR CNRS 5158, Alle Alain Savary, 21000 Dijon, France
3
CLIPP - Clinical and Innovation Proteomic Platform, CGFL - 1 rue du Professeur Marion BP 77980, 21079 Dijon, France Contact: Valentin Bouquet (vbouquet@asa-sas.com)
Keywords: Bioinformatics et biostatistics dedicated to proteomics, Clinical proteomics Lobjectif de la protomique clinique est didentifier de nouveaux biomarqueurs protomiques qui soient diagnostiques ou pronostiques dune pathologie donne. Pour augmenter la pertinence des analyses, les donnes protomiques peuvent tre analyses conjointement aux variables clinico-biologiques classiques. Ces analyses impliquent un volume de donnes important ncessitant une automatisation et une traabilit dun certain nombre de tches. Dans cette perspective, la plateforme protomique CLIPP sest associe avec le laboratoire en informatique LE2I et la socit ASA pour concevoir et mettre en place un pipeline applicatif facilitant la gestion et lanalyse des donnes de spectromtrie de masse depuis la rception des prlvements de patients jusqu la slection de marqueurs pertinents. La premire partie du pipeline a pour objectif de prendre en charge le flux de donnes clinicobiologiques en provenance des cliniciens collaborant avec la plateforme et des Centre de Ressources Biologiques (CRB). La gestion de ce processus implique la mise en place dun systme de gestion du cycle de vie des chantillons et dune base de donnes suffisamment flexible pour accueillir des donnes cliniques htrognes dune pathologie lautre. Le logiciel eClims, dvelopp par Pierre Naubourg (LE2I), prend en charge ces sources de donnes complexes et assure le suivi des prlvements de patients ds leur arrive sur la plateforme tout en normalisant/formatant les informations clinico-biologiques associes. eClims devient la banque de donnes rfrenant lensemble des informations cliniques associes aux prlvements disponibles sur la plateforme. La deuxime partie du pipeline assure le suivi des traitements analytiques subis par les prlvements pour leur analyse en spectromtrie de masse. Le systme ePims, dvelopp par le laboratoire EDyP, a t mis en place sur la plateforme afin de tracer le passage des chantillons sur les instruments, sauvegarder et organiser les acquisitions gnres. Un travail dinterfaage entre eClims et ePims garantit un suivi global des prlvements. Les chantillons enregistrs dans eClims sont automatiquement injects dans ePims, vitant par exemple les erreurs de saisie. Enfin, la troisime partie du pipeline se charge de prtraiter les acquisitions stockes dans ePims et de mettre disposition des statisticiens, une interface leur permettant dextraire les donnes clinico-biologiques ainsi que les donnes protomiques rattaches un projet particulier pour pouvoir ensuite les analyser. Le logiciel eP-Stat, dvelopp par Valentin Bouquet (ASA), assure ces deux dernires tapes en mettant en place, dune part, un processus dexcution automatique (deamon) des scripts R raliss sur la plateforme et dautre part un moteur de recherche puissant capable dextraire les donnes stockes la fois dans eClims et dans ePims. Link: www.asa-sas.com
209/381
[P123] Efficient structural characterization of synthetic polymers by activated electron photo-detachment dissociation
Marion Girod1, Claire Brunet1, Rodolphe Antoine1, Jrme Lemoine3, Philippe Dugourd1, Laurence Charles2
1
Laboratoire de Spectromtrie Ionique et Molculaire, CNRS et Universit Lyon I, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France
2
Laboratoire Chimie Provence, Spectromtries Appliques la Chimie Structurale, Universits Aix-Marseille I, II & III et CNRS, Campus St-Jrme, 13397 Marseille Cedex 20, France
3
Institut des Sciences Analytiques, NRS et Universit Lyon I, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France Contact: Marion Girod (mgirod@lasim.univ-lyon1.fr)
Keywords: Organic and inorganic mass spectrometry, Instrumentation Tandem mass spectrometry of poly(styrene sulfonate sodium salt) (PSS) and poly(methacrylic acid) (PMAA) was performed after activated electron photo-detachment dissociation (activated EPD). In this technique, doubly charged oligomers were first produced in negative mode electrospray ionization, then oxidized into radical anions upon electron photo-detachment using a 220 nm laser wavelength, and further activated by collision. In contrast to CID of negatively charged PSS oligomers, which does not provide informative data with regards to the end-groups, activated-EPD is shown here to promote radical-induced dissociation reactions thanks to the oxidation of a sulfonate group upon laser irradiation. Major product ions generated after backbone bond cleavages contained one or the other chain terminations and could be accounted for by four main mechanisms, depending on the location of the oxidized monomer near one or the other chain terminal moieties. As a result, combination of these two fragments allowed a straightforward mass characterization of each end-group. In case of PMAA ions, fragmentation of odd-electron species is first shown to proceed via a radical-induced decarboxylation, followed by reactions which involved backbone bond cleavages and gave rise to product ions containing one or the other oligomer termination, contrasting with conventional collision induced dissociation of negatively charged PMAA, which mainly consists of multiple dehydration steps. Activated EPD spectra displayed quite intense product ions throughout the whole m/z range, allowing unambiguous and straightforward determination of each end-group mass. Experiments performed using PMAA sodium salts allowed us to account for relative intensities of peaks measured within product ion series obtained from PMAA, which would reveal that ion stability is ensured by hydrogen bonds within pairs of MAA units.
210/381
[P124] Le Couplage CE-ICP/MS : un outil de choix pour lEtude des Interactions Protine-Mtal
Agns Hagge1, Olivier Averseng 1, Claude Vidaud1
1 2
CEA/DSV/SBTN, Laboratoire dEtude des Protines Cibles, Site de Marcoule, 30207 Bagnols-sur-Cze, France UMR 6191 CNRS/ Universit Aix-Marseille/CEA, Cadarache, 13108 Saint Paul lez Durance, France
Contact: Agns Hagge (agnes.hagege@cea.fr)
Keywords: Organic and inorganic mass spectrometry, Separative analysis methods, Instrumentation Aprs internalisation, les mtaux non biologiques comme luranium peuvent se fixer sur diffrents composants cellulaires, notamment les protines et altrer leur fonction. Hormis quelques tudes rcentes[1,2], on connat cependant encore peu de choses sur les interactions protine/uranium qui stablissent et les consquences de ces interactions sur la toxicit, la majorit des tudes tant focalise sur lalbumine et la transferrine.[3-6] La comprhension de ces mcanismes dinteraction est cependant indispensable lanalyse des risques et au dveloppement dagents complexants spcifiques afin dlaborer des stratgies de prvention et de traitement. Dans ce contexte, nous avons mis au point un systme de crible, bas sur le couplage entre llectrophorse capillaire (CE) et la spectromtrie de masse ionisation par plasma (ICP/MS). Son intrt rside dans la possibilit dtudier les interactions protine/mtal partir de mlanges complexes et ceci pour plusieurs raisons. Dune part, les complexes rsultant sont en effet le fruit de nombreuses ractions de comptition, compte tenu de la prsence au sein des divers compartiments biologiques de protines, de lipides, de petits ligands (carbonate, citrate, ..) mais aussi de mtaux physioessentiels. Dautre part, certaines interactions ne peuvent tre simplement dcrites par des modles ne mettant en jeu que deux partenaires. Dans ce cadre, nous montrerons sur diffrents mlanges simples de protines que lutilisation du couplage CE-ICP/MS se rvle tre un outil de choix, permettant une sparation de ces protines dans des conditions peu dnaturantes allie une dtection sensible sans ambigut de luranium mais aussi des mtaux endognes. Grce loptimisation de collisions de haute nergie dans le spectromtre, nous montrerons galement la faisabilit dune dtection du soufre par un spectromtre quadripolaire, bnficiant ainsi dune dtection dune grande partie des protines. 1.Dedieu et al., J. Chromatogr. A, 2009, 1216, 5365. 2.Vidaud et al., J. Chromatogr. A. 2008, 1185, 233. 3.Chevari et al., Med. Radiol.1969, 14, 29. 4.Duff Jr et al., Angew. Chem. 2006, 118, 143. 5.Ansoborlo et al., Biochimie 2006, 88, 1605. 6.Averseng et al., Anal. Chem. 2010, 82, 9797.
211/381
[P125] Etude du phosphoprotome de Pseudomonas aeruginosa

Tassadit Ouidir1, Laurent Coquet1, Adle Bourmaud2, Philippe Chan2, Pascal Cosette1, Thierry Jouenne1, Julie Hardouin1
1 2
Laboratoire PBS - UMR CNRS 6270, Universit de Rouen, 76821 Mont-Saint-Aignan cedex
UMR 6270 CNRS - Plate-forme Protomique de lIFRMP23, Universit de Rouen, 76821 Mont-Saint-Aignan cedex Contact: Julie Hardouin (julie.hardouin@univ-rouen.fr)
Keywords: Signaling, interactomics and post-translational modifications Pseudomonas aeruginosa est un pathogne opportuniste qui merge ces dernires annes comme responsable dinfections nosocomiales svres chez des sujets immunodprims. Cette bactrie pose de relles difficults thrapeutiques en raison de sa capacit former des biofilms. Rcemment, il a t montr au sein de lUMR 6270 CNRS quune dltion du gne pA3731 induit une diminution de sa capacit dadhsion. De nombreux phosphorelais tant impliqus dans les mcanismes dadhsion, nous nous sommes intresss limpact de la mutation de ce gne sur le changement du profil phosphoprotique de P. aeruginosa. Les phosphoprotines sont fortement minoritaires dans le protome total et leur enrichissement est donc ncessaire. Dans cette tude, nous avons utilis diffrentes colonnes daffinit afin denrichir les chantillons en phosphoprotines ou en phosphopeptides. Les protines retenues sur ces colonnes ont t visualises par gel 2D. Aprs digestion trypsique, les identifications ont t ralises par spectromtrie de masse. Des phosphoprotines dj dcrites comme phosphoryles dans la littrature ont t identifies. Un nouveau site de phosphorylation a galement t caractris.
212/381
[P126] Heparin-like disaccharides: effects of metallic complexation fragmentation as characterized by MS/MS and UVPD experiments
Daniel Ortiz1, Quentin Enjalbert2, Jean-Yves Salpin1, Philippe Dugourd2
1
upon
Laboratoire Analyse et Modlisation pour la Biologie et l'Environnement - LAMBE - UMR 8587 - Universit d'Evry Val d'Essonne, Boulevard Franois Mitterrand, 91025 Evry
2
Laboratoire de Spectromtrie Ionique et Molculaire - LASIM - UMR 5579 - Universit Claude Bernrd Lyon 1, Btiment A. Kastler - Boulevard du 11 novembre 1918, 69622 Villeurbanne Contact: Jean-Yves Salpin (jean-yves.salpin@univ-evry.fr)
Keywords: Organic and inorganic mass spectrometry, Instrumentation Heparin (HP) glycosaminoglycan (GAGs), an anticoagulant drug, is recognized to be a biologically important polysaccharide and is implicated in many biological processes [1]. Given the relevance of GAGs, many efforts were devoted in the last years to the characterization of Heparins by mass spectrometry [2]. In previous works, Dugourds group reported the optical spectra and photodissociation patterns of gas-phase deprotonated anions of HP derivatized disaccharides [3-5]. On the other hand, the LAMBE group has already demonstrated that the gas-phase reactivity of metal ions can be particularly helpful in differentiating isomeric saccharides [6-8]. The present work reports the comparison between CID/UVPD modes of three heparin-derived disaccharides complexes. Analytically it is demonstrated the successfully use of metal complexation in order to distinguish all the isomers. Without metal complexation the CID spectra for II-S and III-S are basically the same, but when [M(Hep)-H]- is formed it is observed a significant change in the fragmentation pathway. It is also shown that UVPD leads to different and more informative fragmentation than the CID mode. It is also revealed that there is an unambiguous blue shift effect in the carboxylic group absorption due to the metal complexation. DFT calculations were carried out to in order to account for the observed UV spectra, and to propose different fragmentations pathways for all the mono-sulfated heparins. References 1. J.I. Capila, R.J. linhardt. Angew. Chem. Int. Ed. 41, 391 (2002) 2. E.F. Naggar, C. E. Costello, J. Zaia. J. Am. Soc. Mass Spectrom. 15, 1534 (2004) 3. A. Racaud, R. Antoine, L. Joly, N. Mesplet, P. Dugourd, J. Lemoine. J. Am. Soc. Mass Spectrom. 20, 1645 (2009). 4. A. Racaud, A. R. Allouche, R. Antoine, J. Lemoine and P. Dugourd. J. Mol. Struct. 960, 51 (2010). 5. A. Racaud, R. Antoine, P. Dugourd and J. Lemoine. J. Am. Soc. Mass Spectrom. 21, 2077 (2010). 6. J-Y. Salpin, L. Boutreau, V. Haldys, J. Tortajada. Eur. J. Mass Spectrom. 7, 321 (2001) 7. J-Y. Salpin, J. Tortajada. J. Mass Spectrom. 37, 379 (2002). 8. A. El Fidoussi, M.Lafitte, J. Tortajada, O. Kone, J-Y. Salpin. J. Mass Spectrom. 42, 999 (2007)
213/381
[P127] Measurement of vitellogenin protein in invertebrates: relevance and usefulness of mass spectrometry (LC-MS/MS) to propose a specific and transferable method across species
Romain Simon1, Fabien Audouard-Combe2, Guillaume Jubeaux2, Renaud Tutundjian2, Herv Quau2, Jeanne Garric2, Olivier Geffard2, Jrme Lemoine1, Arnaud Chaumot2, Arnaud Salvador1
1 2
Institut des Sciences Analytiques, Universit de Lyon, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Unit de Recherche MALY, Cemagref Lyon, 3 B Quai Chauveau, 69009 Lyon
Contact: Romain Simon (rom1.simon@orange.fr)
Keywords: Proteins, peptides and small molecules quantification Reproductive success of organisms is related to the quantity and quality of eggs produced by females. Vitellogenin (Vg) is the precursor protein of vitellin that is the energy available for embryonic development in oviparous organisms. Vg is proposed as a relevant biomarker of endocrine disruptors in aquatic vertebrate species (e.g. fish, amphibian). Numerous strategies, such as enzyme-linked immunosorbent assays have been developed to characterize and quantify this protein in vertebrates. On the contrary, in invertebrates few methods are available in spite of their importance. This gap mainly comes from the low transferability of available antibodies across species. Recently, our laboratory developed a quantitative assay of Vg in a widespread amphipod, Gammarus fossarum, using liquid chromatography tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode (SRM). In this context, the aim of this study was to assess the possibility to take advantage of peptidic motifs conservation to propose a transferable method across species. For this, our study focused on three model species for which sequence of egg yolk protein is well known: an amphipod (Gammarus fossarum), a cladocerean (Daphnia magna) and an insect (Drosophila melanogaster). In a first step, proteotypic peptides of Vg were identified for each species. Then we tried to find these proteotypic peptides in closely related species for which Vg sequence is unknown (such as G. pulex, G. wautierii, D. pulex). This study showed the high relevance of mass spectrometry to propose a specific methodology for Vg absolute quantification in organisms for which proteins sequences are unknown. Moreover, the proposed methodology is transferable from a species to another one.
214/381
[P128] 2D ECD/IRMPD FT-ICR MS Analysis of Glycopeptides

Maria A. van Agthoven1, Marie-Aude Coutouly3, Marc-Andr Delsuc2, Christian Rolando1
1
USR 3290 MSAP, Universit Lille 1, Sciences et Technologies, bt C4, Cit Scientifique, 59655 Villeneuve d'Ascq cedex
2
Institut de Gntique et de Biologie Molculaire et Cellulaire, INSERM, U596; CNRS, UMR7104; Unversit de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch-Graffenstaden
3
NMRTEC, Bld. Sbastien Brandt, Bioparc - Bat. B, 67400 Illkirch-Graffenstaden
Contact: Maria A. van Agthoven (marie.van-agthoven@univ-lille1.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications Glycosylation is a common post-translational modification in proteins. In 2001, Hkansson et al. showed that Electron Capture Dissociation (ECD) and Infrared MultiPhoton Dissociation (IRMPD) are fragmentation techniques that yield complementary information on the structure of tryptic digest glycopeptides: IRMPD preferentially cleaves the glycosidic bond, while ECD cleaves the peptide backbone while leaving the glycosidic bond intact [1]. Since both IRMPD and ECD are commonly available on commercial FT-ICR mass spectrometers, determining the amino acid sequence of the glycopeptide, the position of the glycosylation site and the nature of the glycan by recording MS/MS spectra of glycopeptides is easy. We have successfully implemented 2D FT-ICR MS pulse sequences using both ECD and IRMPD on a commercial Bruker Daltonics 9.4 T ApexQE FT-ICR mass spectrometer [2]. This technique allows us to correlate the parent and fragment ions of a complex sample without isolating the various ion species before fragmenting them. We have also successfully reduced the signal-tonoise ratio by applying the Cadzow algorithm to 2D mass spectra [3] and we are now able to record and process spectra with high horizontal resolution and quadrupole-like vertical resolution. This enables us to use the fragmentation patterns of peptides to determine their amino acid sequence. By combining ECD and IRMPD we can also locate and identify possible glycosylation sites in peptide mixtures like tryptic digests. In this study, we used custom-made synthetic O-glycosylated peptides (SVES(-OGlcNAc)GSADAK-NH2, SVET(-O-GlcNAc)GSADAK-NH2, YSPTS(-O-GlcNAc)PSK-NH2) in order to optimize the determination of amino acid sequences and the location of glycosylation sites with ECD. In order to identify glycans using IRMPD, we used glycopeptides with more complex sugars, like vancomycin and bleomycin. We show the 2D ECD and IRMPD mass spectra of these compounds in mixtures, the sequence coverage we obtain from them and the identification of the nature of the glycosylation. We show that, in 2D FT-ICR MS, ECD and IRMPD are complementary tools not just for one, but for a mixture of glycopeptides and peptides for the complete identification of proteins and their glycosylations. The combination of the two fragmentation modes therefore has the potential to greatly facilitate the identification of post-translational modifications in proteomics. [1] Hkansson et al. Anal. Chem. (2001) 73, 4530-4536. [2] M.A. van Agthoven et al. Int. J. Mass Spectrom. (2010) http://dx.doi.org/10.1016/j.ijms.2010.10.034. [3] M.A. van Agthoven et al. Rapid Commun. in Mass Spectrom. (2011) 25, 1609-1616.
215/381
[P129] Etude de la glycosylation danticorps produits en milieu sans srum

Jonlet Pierre1, Nicolas Smargiasso1, Sophie Waxweiler2, Alfred Collard2, Edwin De Pauw1
1 2
Laboratoire de Spectromtrie de Masse (LSM), Universit de Lige, 3 Alle de la Chimie, 4000 Lige, Belgique CER Groupe- Dept Biotechnologie, Plateforme culture cellulaire,, 1 Rue du Carmel, 6900 Marloie, Belgique
Contact: Jonlet Pierre (pjonlet@ulg.ac.be)
Keywords: Clinical proteomics, Signaling, interactomics and post-translational modifications Les chaines glycosyles jouent un rle important dans lactivit biologique des anticorps (Ac) produits de manire recombinante et leur profil oligosaccharidique est dpendant des conditions de culture. Afin doptimaliser la production de longue dure danticorps monoclonaux dans des cellules cultives en milieu sans srum et dobtenir des produits conformes, il est important de caractriser la glycosylation de ces protines par rapport aux protines natives, et cela en fonction du milieu utilis et de la dure des cultures. Le suivi de ce paramtre permet dassurer une production maximale tout en conservant les caractristiques de la protine produite, un mauvais profil de glycosylation pouvant engendrer une absence de reconnaissance de lantigne. Dans ce contexte, quatre milieux chimiquement dfinis ont t choisis pour tester la production de ces protines. La part de srum dans le milieu de culture a t diminue progressivement afin de permettre ladaptation des cellules un milieu sans srum. Notre tude porte sur lanalyse de changements potentiels dans le profil de glycosylation intervenant au cours de ce processus dadaptation. La premire approche a consist tudier les protines entires (ici les chaines lourdes des Ac) par ESI-Q-TOF afin dobtenir rapidement les profils de glycosylation prsents. Par la suite, nous nous sommes focaliss sur certains milieux afin de dterminer plus prcisment les structures des glycanes prsents. Dans ce cas, les glycanes ont t extraits, permthyls puis analyss par MALDI-TOF. Des expriences de MS/MS ont de plus permis de mettre en vidence certaines structures prsentes. Enfin, la localisation de la partie glycosyle au sein de la protine a t ralise laide de la spectromtrie de masse en tandem CID-ETD. En combinant les premiers rsultats obtenus, il apparait que la glycosylation des protines produites grce aux milieux sans srum est globalement proche de celle obtenue pour les protines produite de manire traditionnelle. Les espces principales sont en effet identiques. De plus, la fucosylation est stable dans les diffrents chantillons analyss (80 90% des glycanes contiennent un fucose). Nanmoins, certaines diffrences peuvent apparatre concernant la prsence et/ou la nature des acides sialiques rtrouvs. En ce qui concerne la localisation des parties glycosyles, les rsultats obtenus montrent quil ny a quun seul site portant les glycanes et que celui-ci reste identique dans les diffrentes conditions testes.
216/381
[P130] Drivation chimique de phosphopeptides basiques comprenant une cystine pour amliorer leur dtection par spectromtrie de masse
Lucrce Matheron1, Oumar Diebate1, Philippe Karoyan1, Emmanuelle Sachon1, Dominique Guianvarc'h1, Grard Bolbach1
1 2 3
Laboratoire des Biomolcules, UMR 7203 CNRS-UPMC-ENS, 4 place Jussieu, 75005 Paris, France Plateforme de spectromtrie de masse et protomique, IFR83, 7-9 quai saint Bernard, 75005 Paris, France
Laboratoire des Biomolcules, UMR 7203 CNRS-UPMC-ENS, Ecole normale suprieure, dpartement de chimie, 24 rue Lhomond, 75005 Paris, France Contact: Lucrce Matheron (lucrece.matheron@gmail.com)
Keywords: Signaling, interactomics and post-translational modifications La dtection des phosphopeptides par spectromtrie de masse (MS) reste difficile cause i) du faible rendement dionisation de ces espces, ii) des phosphorylations in vivo non totales, et iii) des effets de suppression dans les mlanges. Lune des mthodes employes pour pallier ces difficults est la purification slective des espces phosphoryles. Cependant, les mthodes majeures de chromatographie daffinit sont peu efficaces pour les phosphopeptides avec de multiples rsidus basiques. Nous avons pu le vrifier sur des substrats de Protine Kinase C, dont la squence de base est CFRTPSFLKK, phosphoryls sur la Ser et contenant 3 rsidus basiques. Ces phosphopeptides ont une trs faible affinit pour les matriaux classiques denrichissement. Nous avons donc dvelopp une mthodologie de drivation chimique pour en amliorer lanalyse par MALDI-TOF, en particulier en mlange avec les peptides non phosphoryls correspondants. La drivation chimique , en conditions basiques, se ralise en deux tapes : (i) -limination du phosphate et (ii) addition de Michael sur la double liaison ainsi forme pour ajouter une fonction chimique favorable lionisation. Lors de la -limination, nous observons une forte perte de SH2 sur la cystine. Llimination du phosphate et les ractions secondaires ntant pas totales, on obtient de nombreux produits, ce qui complique les spectres MALDI-TOF et surtout diminue lintensit des signaux observables. Jouer sur la cintique et la temprature permettent de diminuer ces ractions secondaires, mais au dtriment de llimination du phosphate. Nous cherchons modifier la base utilise pour obtenir une limination totale sur la pSer et sur la Cys, ce qui conduirait un produit unique et une double addition de Michael. Divers nuclophiles ont t tests comme agents de drivation sur des mlanges quimolaires peptide phosphoryl et non phosphoryl, en particulier le mercaptothylpyridine (MEP), la thiocholine et le 2-phnylthanethiol. Alors quavant drivation, les phosphopeptides sionisent 3 5 fois moins bien en MALDI-TOF, la drivation par le MEP permet dobtenir des intensits quivalentes. La thiocholine donne lieu des fragmentations en MALDI-TOF, qui diluent le signal obtenu. Enfin, la drivation par le 2phnylthanethiol permet dobtenir des intensits pour le peptide driv pouvant tre trs suprieures celle du peptide non phosphoryl. Finalement, la limite de dtection de la mthode sera prsente, sous deux aspects : (1) Pour un mlange quimolaire, quelle est la limite en quantit totale de peptide ? (2) quantit de peptide totale fixe, quel est lexcs maximal de peptide non phosphoryl permettant de continuer observer le peptide driv ?
217/381
[P131] The mechanisms of fat loss during cooking of foie gras: Proteomic analysis of duck fatty liver
Laetitia Thron1, Carole Pichereaux2, Caroline Molette1, Nathalie Marty-Gasset1, Christophe Chambon4, Michel Rossignol2, Didier Viala4, Xavier Fernandez1
1 2
INRA, INPT-ENSAT, INPT-ENVT, UMR 1289 TANDEM, Avenue de l'Agrobiopole, 31326 Castanet-Tolosan
IPBS-FR3450, Plateforme Protomique du Gnopole Toulouse Midi-Pyrnes, 205 route de Narbonne, 31077 Toulouse
3 4
Universit Paul Sabatier, Universit de Toulouse, route de Narbonne, 31077 Toulouse
Plateforme dexploration du Mtabolisme : Composante Protomique, INRA, UR370, INRA de theix, 63122 Saint Gens-Champanelle Contact: Laetitia Thron (laetitia.theron@ensat.fr)
Keywords: Systems biology Fat loss during cooking of duck foie gras is the main quality issue in the industry. To better understand the biological mechanisms involved in this phenomenon, a proteomic analysis was conducted on raw livers. The aim was to characterize changes in protein expression early post mortem (pm) and after chilling, according to fat loss during later cooking. The proteomic analysis of duck foie gras was performed on two protein fractions. The soluble fraction at low ionic strength (SF) was analyzed by two-dimensional electrophoresis and the spots of interest were identified by mass spectrometry both MALDI-TOF and LC-MS/MS. The non soluble fraction at low ionic strength (NSF) was analyzed by a shot-gun approach combining a separation by SDS-PAGE and identification by a tandem liquid chromatography-mass spectrometry system (nano-HPLC coupled to an Orbitrap-XL). Quantitative analysis of the results indicated that the fatty livers which showed a low fat loss during cooking exhibited a higher metabolic activity early pm (i.e. in the SF, the glycolytic enzymes G3PDH, alpha enolase and triose phosphate isomerase spots intensities were higher). In the "NSF" fraction, 70 proteins were only present in the sample with a low-fat loss and, according to the Gene Ontology database, the proteins implicated in the translation activity were the most abundant. The fatty livers which showed a high fat loss during cooking exhibited a higher expression of proteins involved in some protective mechanisms of cells. The two main proteins involved were HSP 27 and Calponin-1, a calcium binding protein which expression is linked to the activation of activate stellate cells and chronic liver injury (Feng et al., 2005). After the chilling, the fatty livers showed an increasing expression of proteins from the cytoskeleton. This is most likely due to the appearance of proteins fragments rather to protein synthesis which is unlikely in the post mortem tissue. We draw the hypothesis that the structure of the tissue would be thus more fragile and prone to fat loss during cooking. The proteomic analysis allowed us to hypothesize the scheme of evolution of fatty liver from slaughter to chilling, in relation with the fat loss during cooking. Further studies should investigate the proteolysis in relation to fat loss during cooking.
218/381
[P132] GC, GC-MS and GC-GC-MS analysis of essential oils isolated from Nigella sativa seeds by accelerated techniques assisted by microwaves using the cryogenic grinding
Farid Benkaci-Ali1, Edwin De Pau2, Rym Akloul1, Asma Boukenouche1
1
Universit des Sciences et Technologies Houari Boumediene, Facult de Chimie, Laboratoire dAnalyse Organique Fonctionnelle, , B.P. 32 El Alia, Bab Ezzouar, Algrie, 16001 Alger
2
University of Lige, Laboratoire de Spectromtrie de Masse L.S.M, , Alle du 6 Aot, Bt B6c, 4000 Lige (Sart-Tilman), Belgium, Lige Contact: Farid Benkaci-Ali (benkaciaf@yahoo.fr)
Keywords: Systems biology Application of green technologies as cryogrinding and microwaves in extraction methods is considered highly promising since these techniques could improve safety and quality of natural product. The increasing interest of nutritional and pharmacological power of Nigella sativa seeds by products, particularly at industrial level, had motivated us to investigate the chemical content of Nigella sativa essential oil. Extraction of essential oil from Nigella sativa seed treated by cryogrinding (CG) has been conducted by three different procedures: conventional hydrodstillation (HD), steam distillation assisted by microwaves (SDAM) and steam distillation assisted by microwaves with cryogrinding (SDAM-CG). The SDAM and SDAM-CG essential oils were found to exhibit high level in active product as thymoquinone THQ (57%, 43.05% respectively) compared to HD technique (22,4%). The conversion rate (CR) of THQ to thymohydroquinone under heat effect is also very lower for SDAM and SDAM-CG (0.89%) and SDAM-CG (0.96%) compared to the HD procedure (55%). In addition the SDAM-CG technique has provided high yield content (1.3 %) compared to SDAM (0.9%) and HD-CG (0.34%). The composition of the volatile oil has been investigated by capillary gas chromatography (GC) and gas chromatography-mass spectrometry GC-MS. The cryogrinding coupled to microwaves and steam distillation process (SDAM and SDAM-CG) constitute the adequate technique to extraction operations from the yields and the high content in major component, and allows minimizing the energy consumption (95 % compared to HD), the heating time (10 mn), the formation of artefact products (thymohydroquinone and dithymoquinone). So, its profitable to treat some plants and seeds using this process for preserve their thermolabile components.
219/381
[P133] The use of immobilized trypsin bioreactors with MALDI TOF/TOF for protein digestion and identification studies
Raul NICOLI1, Sylvie MICHELLAND2, Morgane COUVET2, Serge RUDAZ1, Jean-Luc VEUTHEY1, Michel SEVE2
1
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, bvd dYvoy 20, 1211 Geneva, Switzerland
2
Plate-forme Protomique Promthe, CR INSERM/UJF U823, Institut Albert Bonniot, IBP CHU Grenoble BP217, 38043 Grenoble, France Contact: Michel SEVE (Michel.Seve@ujf-grenoble.fr)
Keywords: Systems biology Trypsin is the most widely used proteolytic enzyme for protein digestion, which is classically performed in-solution with incubation protocols of several hours. In the development of rapid and automated analytical techniques, an attractive alternative is the use of solid-phase protein digestion through the immobilization of trypsin on monolithic materials (e.g. methacrylatebased disks). With this strategy, flow-through protein digestion can be achieved on-line with the peptides separation and detection by inserting an immobilized trypsin bioreactor in a LC-ESIMS/MS system. In this work, an analytical approach was developed involving flow-through protein digestion in an immobilized trypsin bioreactor and off-line MALDI-TOF/TOF peptides analysis. Optimization of this process in terms of digestion flow rate, amount of digested protein(s) and use of a trapping column was carried out with beta-lactoglobulin containing samples. This strategy was then applied to the digestion of a more complex sample of five proteins with the integration in the analytical set-up of reversed-phase nano-LC peptides separation and automated MALDI spotting device. The results obtained throughout the study were compared with those of classical in-solution incubation method.
220/381
[P134] Dveloppement dune mthode danalyse des Retardateurs de Flammes Broms, plus particulirement de lHexabromocyclododcane (HBCD) par chromatographie liquide couple la spectromtrie de masse en tandem (LCESI-MS-MS)
Zita Zendong1, Cyrille Valle1, Frederic Larvor1, Sophie Durand1, Philippe Marchand1, Jean-Philippe Antignac1, Bruno Le Bizec1
1 2
Laboratoire d'etudes des residus et contaminants, Route de gachet BP 50707, 44307 Nantes INRA NANTES, ,
Contact: Zita Zendong (zita.zendong@oniris-nantes.fr)
Keywords: Systems biology Les Retardateurs de Flammes Broms (RFB) ont t largement utiliss dans divers produits commerciaux dans le but de rduire limportance des incendies. Le 1,2,5,6,9,10Hexabromocyclododcane (HBCD) est le troisime retardateur de flamme brom le plus utilis dans le monde (16700 tonnes en 2001) et le deuxime plus utilis en Europe. LHBCD est obtenu par bromation du cyclododca-1,5,9-trine et se compose principalement de trois paires de diastroisomres : alpha, beta et gamma-HBCD, le gamma-HBCD tant majoritaire. Ces diffrents diastroisomres ont des proprits diffrentes linstar de leur solubilit dans leau qui est respectivement de 48,8, 14,7 et 2,1 g/L pour les alpha, beta et gamma-HBCD. Comme tous les autres retardateurs de flammes broms, lHexabromocyclododcane (HBCD) peut tre relch dans lenvironnement via les missions durant sa production ou par des fuites suite lutilisation des produits traits avec ce dernier. Faiblement biodgradable et avec un coefficient de partage octanol/eau (Kow) lev, lHBCD a un potentiel lev de bioaccumulation dans les tissus adipeux dorganismes vivants. Par ailleurs sa toxicit et sa persistance dans lenvironnement pouvant conduire des dommages irrversibles, ce compos suscite des inquitudes en tant que potentiel contaminant. Do la ncessit de mettre au point des mthodes danalyses dans le but de le quantifier de manire prcise. La plupart des donnes environnementales publies dernirement ont t obtenues en utilisant la spectromtrie de masse couple la chromatographie gazeuse. Cependant les isomres de lHBCD ntant pas sparables sur les colonnes conventionnelles de chromatographie gazeuse, seule la somme de tous les isomres de lHBCD tait quantifie. Une nouvelle mthode utilisant la chromatographie liquide couple la spectromtrie de masse en tandem (LC-ESI-MS-MS) a donc t dveloppe pour quantifier sparment les isomres alpha, beta et gamma de lHBCD. Cette mthode applicable des matrices varies consiste en une extraction liquide sous pression (ASE) suivie de purifications sur phases solides lissue desquelles lextrait obtenu est repris dans le mthanol, solvant permettant une plus grande stabilit des isomres dHBCD. Llution de ces composs seffectue ensuite sur une colonne Hypersil Gold (1,9m ; 100 mm ; 2,1 mm) avec comme solvants un mlange mthanol/Eau (50:50, v:v) et de lactate dammonium 20mM. Lidentification de chaque isomre se fait sur la base de deux ions du massif isotopique caractristiques de lion pseudomolculaire. Cette mthode permet la sparation des trois diastroisomres en 8 minutes.
221/381
[P135] Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulinreleasing activity.
Milena Mechkarska1, Opeolu O. Ojo2, Mohammed A. Meetani 3, Laurent Coquet4, Thierry Jouenne4, Yasser H.A. Abdel-Wahab2, Peter R. Flatt2, Jay D. King5, J. Michael Conlon1
1
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates
2 3
School of Biomedical Sciences, University of Ulster, Cromore Road, BT2 ISA Coleraine, North Ireland, UK
Departement of Chemistry, Faculty of Science, United Arab Emirates University, 14551 Al-Ain, United Arab Emirates
4
CNRS UMR6270, European Institute for Peptide research, University of Rouen, 76821 Mont-Saint-Aignan, France
5
Rare Species Conservatory Foundation, , MO 63110 Saint Louis, USA
Contact: Laurent Coquet (laurent.coquet@univ-rouen.fr)
Keywords: Clinical proteomics Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus (Shaw, 1802) led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal Beta-cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (236% of basal rate) at a concentration of 3M. Ranatuerin2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 M, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb(FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate) but the peptide was cytotoxic at this concentration.
222/381
[P136] Mass Spectrometry Imaging visualization tools developed during the Computis European project
Marie-France Robbe1, Etienne Thvenot1, Markus Stoeckli2, Alfons Hester3, Andreas Roempp3, Andriy Kharchenko4, Olivier Gal1, Serge Haan1
1
CEA Saclay, LIST, Laboratoire d'Outils d'Analyses de Donnes, Btiment 516, F-91191 Gif sur Yvette cedex, France
2 3 4
Novartis, Lichstrasse 35, WSJ-507.1101, CH-4002 Basel, Switzerland Justus Liebig University, Schubertstrasse 60, Building 16, D-35392 Giessen, Germany FOM/AMOLF, Kruislaan 407, NL-1098 SJ Amsterdam, The Netherlands
Contact: Marie-France Robbe (marie-france.robbe@cea.fr)
Keywords: Imaging mass spectrometry The Computis European project (2006-2009) was aimed at developing innovative experimental mass spectrometry imaging (MSI) techniques and software tools for data treatment and visualization, and at validating them in key biological applications (neurobiology, pharmaceutical drug development). The project succeeded in defining a specific standard format for MSI, imzML, in collaboration with the HUPO consortium. The four main MSI software tools developed by the project all handle the imzML format. Data Cube Explorer provides an easy spectral and spatial exploration of MS images: spectrum zooming, scrolling through the dataset masses with a manual contrast tuning for images, selection of Regions Of Interest with the display of the associated spectra. The self-organizing map functionality classifies images according to the intensity of all pixel places and automatically selects images as different as possible. Mirion is a simple visualization module displaying spectra by pixel and for the total image, with zooming and scrolling functions. Histogram of the total ion count of each pixel can be calculated, using different input parameters. Images are displayed for each peak of the total spectrum, with a manual intensity tuning and a comparison of the intensity distributions by pixel between several images. EasyMSI enables spatial and spectral visualization of mass spectrometry imaging datasets (spectrum and image display, peak and pixel picking, zooming on spectra and images, ROI selection), as well as an assistance for the interpretation of data: This assistance includes indicators (relative variance, Moran index, m/z correlation) to highlight peaks that bring interesting information, peak list for molecule identification, spectrum denoising or structure analysis by clustering methods (K-means, fuzzy, hierarchical clustering, diffusion map). EasyMSI offers the advantage of processing and displaying the original data (i.e. without binning). BioMap is an image analysis platform for MSI and Magnetic Resonance Imaging. It includes viewing functions (spectrum and image display, intensity adjustment, zoom, treatment of multiple ROIs, geometrical transformations and operations), and spectrum treatment (spatial or temporal filtering, baseline correction, detrending). More elaborated functions enable a simultaneous view of all dataset images, creation of a movie, statistical and histogram analysis, co-registration of images of one or two dataset(s), and realignment of images. The use and capacities of these tools are presented through a comparative analysis of a rodent urinary bladder dataset in imzML format.
223/381
[P137] PREDALGO, a new multi-subcellular localization prediction tool dedicated to Algae

Marianne Tardif1, Ariane Atteia2, Olivier Vallon4, Michael Specht5, Sabine Brugire1, Norbert Rolland2, Myriam Ferro1, Christophe Bruley1, Gilles Peltier3, Laurent Cournac3
1 2 3
Biologie Grande Echelle, CEA/INSERM/UJF, 17 rue des martyrs , F-38054 Grenoble, France Physiologie Cellulaire et Vgtale, CEA/CNRS/UJF/INRA, 17 rue des martyrs , F-38054 Grenoble, France
Bionergtique et Biotechnologie des Bactries et Microalgues, CEA/CNRS/Aix-Marseille Univ., , F-13108 Saint-Paul-lez-Durance, France
4
Institut de Biologie Physico-Chimique, CNRS/Univ. Pierre et Marie Curie, 13 rue Pierre et Marie Curie, 75005 Paris, France
5
Institute of Plant Biology and Biotechnology, University of Mnster, Hindenburgplatz 55, 48143 Mnster, Germany Contact: Marianne Tardif (marianne.tardif@cea.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics With the growing economical interest in engineering production of energy molecules (hydrogen, lipids and carbohydrates), proteomics efforts were made to better understand the biogenesis and metabolism of the chloroplast of green Algae organisms. The unicellar green algae Chlamydomonas reinhardtii appeared as a model of choice. A crucial issue is to assign the correct subcellular localization to proteins identified in proteome surveys, a task usually assisted by Plant-dedicated localization-predicting softwares (e.g. TargetP). However, when applied to Algae sequences, these softwares tend to missort chloroplast-localized proteins to mitochondria, leading to very poor sensitivity for the chloroplast (40% for TargetP). We thus developped an Algae-specific predictor called PredAlgo. PredAlgo relies on Neural Network programming and uses as biological information the presence of any of the cleavable N-terminal presequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). The challenge was to identify new cTP/mTP in C. reinhardtii in order to train our predictor with algal-specific sets. This was achieved by screening MS/MS spectra generated by organella-specific proteomic studies [1, 2] for semi-trypsic peptides located within the first 100 amino acids of the precursor sequences. These peptide-matches likely represent N-terminal peptides of mature proteins. The data were completed with i) proteins whose cleavage site was previously determined (Edman sequencing), ii) proteins of the secretory pathway with annotated sequence cleavage position (Uniprot datamining) and iii) uncleaved cytosolic proteins. PredAlgo was trained using these C. reinhardtii-specific sets and its performance was evaluated on independant sets of C. reinhardtii sequences. Our predictor showed a highly improved discrimination capacity between chloroplast- and mitochondria-localized proteins, with high sensitivity for the chloroplast (85%). PredAlgo appeared eligible for usage on the closely related algae Volvox carteri. PredAlgo assigned about 20% of the C. reinhardtii proteome (~3000 proteins) to the chloroplast, recovering 64% of an experimental chloroplast proteome previously established by MS/MS survey [2]. In addition, PredAlgo identified ~2400 "chloroplastic" proteins which were absent from the experimental MS proteome and deserved further examination. 1. Atteia, A., et al., Mol Biol Evol, 2009. 26(7): p. 1533-48. 2. Terashima, M., et al., Mol Cell Proteomics, 2010. 9(7): p. 1514-32.
224/381
[P138] Comment, et pourquoi en ESI, le groupement phosphate conduit la formation de liaisons fortes au sein de dimres du type Nuc/Nuc, Nuc/Pept et Pept/Pept ?
Jean-Claude Tabet1, Viet Nguyen1, Bessem Brahim1, Baiyie Xue1, Ying Xu1, Ludovic Muller1, Alice Delvolv1, Sandra Alves1, Carlos Afonso1
1
LSCOB-IPCM Universit Pie, 4 place Jussieu, 75 252 Paris
Contact: Jean-Claude Tabet (jean-claude.tabet@upmc.fr)
Keywords: High mass and high resolution mass Spectrometry, Systems biology Depuis lavnement de mthodes de dsorption douce, comme les modes varis dionisation ambiante, ltude des systmes non-covalents sest dveloppe, quils sagissent de complexes cationiss ou non, de faible ou forte taille, de nature et de proprits diffrentes ou non. Ces systmes peuvent tre prforms en solution (ou dans les gouttelettes) ou non, dan ce dernier cas il ne sagit pas de systmes spcifiques. Dans les autres cas, ils peuvent ltre ou non et leur utilisation du point de vue analytique mrite une attention particulire pour ne par tirer des conclusions htives et errones. Dans tous les cas, ses complexes simplement ou multichargs, positifs ou ngatifs, libres de solvant produits aprs dsolvatation dagrgats chargs dsorbs en phase gazeuse, ncessitent, pour survivre aux processus collisionnels, et dlimination de molcules de solvant, de renforcer leurs tats de charges locaux ou de changer leur nature. Dune autre manire, cela peut tre associ des modifications conformationnelles permettant de rapprocher des sites dtats de charges opposs, ou favoriser les interactions ion-diple. Trs souvent, les interactions hydrophobes existantes en solution, sont plus ou moins mmorises dans les formes dsorbes charges par le jeu de modification dtats de charge locaux. Quelques soit les interactions produites et renforces, le contrle thermo-cintique joue un rle indniable sur la prsence soit de pont salin ou de liaison hydrogne. Ce comportement implique lexistence dun ou plusieurs de formes de type zwitterion cot de types canoniques. Afin dillustrer lensemble ses points, des exemples de complexes non covalent, outre ceux de la mthode de Cooks, seront prsents. Leur comportement sera analys avec les tandems LTQ/Orbitrap et hQH/FTICR en activant les ions avec processus dexcitations ergodiques ou non comme : (i) CID, SORI-CID, HCD, IRMP et (ii) EXD (ECD, EDD, EID, ETD). Ainsi, la discussion sera menes partir de systmes tels que : (i) peptides/phosphopeptide, (ii) dimres de mono nuclotides, (iii) ss, dsDNA/peptide, (iii) DNA/drug, (iv) ss, dsDNA/PNA pour montrer le comportement gnral quant lorigine de leurs stabilits en phase gazeuse indpendamment de leurs charge et leur polarit nette. Nos remerciements vont A. Woods pour ses fructueuses discussions, la participation de maters 2 (S. Trvisiol, de Nguyen N.L.), la SANOFI, la plateforme SM3P, lUPMC et au CNRS.
225/381
[P139] Optimization of Label-Free analysis conditions for the comprehension of Pseudomonas aeruginosa biofilm establishment
Anthony Cotton1, Stphane Claverol1, Sbastien Vilain2, Bertrand Garbay2, Marc Bonneu1
1 2
Plateforme Protome, 146 rue Lo Saignat, 33076 BORDEAUX Cedex
cole Nationale Suprieure de Technologie des Biomolcules de Bordeaux, 146 rue Lo Saignat, 33076 BORDEAUX Cedex Contact: Stphane Claverol (stephane.claverol@u-bordeaux2.fr)
Keywords: Proteins, peptides and small molecules quantification The bacteria immobilized within a biofilm are characterized by an exceptional resistance to the environmental stresses. The exact origin of this resistance is still debated. Among the hypothesis advanced to explain this phenotype; one finds the occurrence of a "biofilm" phenotype among all or part of the immobilized bacteria. Its medical implication; in particular in cystic fibrosis; be the reason for numerous analyses of the modifications of the genome expression of Pseudomonas aeruginosa induced by the biofilm mode of growth. Beside more historical quantitation technique such as bidimensionnal electrophoresis followed by image analysis or mass spectrometry after isotope labeling (i.e. iTRAQ), we have explored the capabilities of label-free mass spectrometry technique for the study of protein whose expression is altered in biofilms compared to planktonic growth conditions. Quantitations were performed on a LTQ-Orbitrap XL mass spectrometer and data were processed using Progenesis LC-MS label-free software. General considerations and optimized parameters relative to the methodology will be presented. Results from sample fractionated or not on SDS-PAGE will be compared. Finally, integration of label-free results with results from other quantitation techniques already performed in our laboratory will be introduced.
226/381
[P140] Aedes caspius mosquito bites: Salivary gland protein repertoire analysis and identification of specific individual exposure markers
Albin Fontaine1, Ibrahima Diouf1, Aurlie Pascual1, Nawal Bakkali1, Luc Camoin2, Emilie Baudelet2, Stphane Audebert2, Franck Remoue3, Frderic Pags4, Lionel Almeras1
1
Unit de Parasitologie, Antenne Marseille de lInstitut de Recherche Biomdicale des Armes (IRBA), Le Pharo, BP 60109,, 13 262 Marseille, France
2 3
INSERM U891, Centre de Recherche en Cancrologie de Marseille, , , 13009 Marseille, France
UR016 Institut de Recherche pour le Dveloppement (IRD), Caractrisation et Contrle des Populations de vecteurs, , 911 avenue Agropolis, BP 64501,, 34394 Montpellier cedex 5, France
4
Unit dEntomologie Mdicale, Antenne Marseille de lInstitut de Recherche Biomdicale des Armes (IRBA), Le Pharo, BP 60109, 13 262 Marseille Cedex 07, France Contact: Lionel Almeras (almerasl@imtssa.fr)
Keywords: Systems biology, Separative analysis methods, Clinical proteomics Mosquito-borne diseases are a major health problem worldwide. Serological responses against mosquito saliva proteins may be useful in estimating an individuals exposure to arthropod vectors. The aims of this study were to determine whether the IgG response level is related to mosquito density, to assess the genus and/or species specificity of this response and to identify corresponding salivary gland antigens. The antibody levels of southeast French individuals living in three areas with distinct Aedes caspius mosquito density were compared by ELISA on several mosquito salivary gland extracts. A significant increase in the antibody responses was observed based on seasonal and spatial Ae. caspius density, and the antibody responses seemed to be specific to the mosquito genus and species. Secreted Ae. caspius salivary proteins were analyzed using in-gel and off-gel methods allowing the identification of more than one thousand proteins including around two hundreds putative secreted salivary proteins. Additionally, antigenic salivary proteins eliciting this specific antibody response were characterized using an immunoproteomic approach. Such antigenic candidates will be used to assess Ae. caspius exposure bites at the individual level, and will serve to evaluate efficiency of mosquito control strategies.
227/381
[P141] Proteomic analysis of Endothelial Colony-Forming Cells (ECFCs) following treatment with Osteoprotegerin (OPG), a new actor in vasculogenesis
Zahia Benslimane-Ahmim2, Florence Poirier1, Dominique Heyman4, Catherine Boisson-Vidal2, Didier Lutomski1
1 2 3 4 5
Universit Paris 13, UMR CNRS 7244 CSPBAT/LBPS, 74 rue Marcel Cachin, 93017 Bobigny, France INSERM, U765, , 75006 Paris, France Universit Paris Descartes, Facult de Pharmacie, , 75006 Paris, France INSERM UMR-S 957, , 44035 Nantes, France Universit de Nantes, , 44035 Nantes, France
Contact: Florence Poirier (florence.poirier@univ-paris13.fr)
Keywords: Signaling, interactomics and post-translational modifications, Systems biology Angiogenesis is a vital mechanism in the bodys response to wound healing and is involved in several pathological processes. Its precisely regulated by a variety of inter- and extra-cellular signals. Osteoprotegerin (OPG) a soluble tumor necrosis factor receptor family molecule which potently inhibits RANKL-mediated osteoclastogenesis, emerges as a new actor in vasculogenesis. It protects mature endothelial cells from apoptosis in vitro and promotes neovascularization ex vivo. We previously found that OPG stimulated migration, chemotaxis and vascular cord formation on Matrigel of Endothelial Colony-Forming Cells (ECFCs). In the present work, we attempted to determine, using proteomics, the changes in protein expression profiles following a treatment of ECFCs with OPG. The identity of differentially expressed proteins was determined by liquid chromatography-tandem mass spectrometry, and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways. This would allow us to obtain a better knowledge of the metabolic pathways affected by OPG treatment and partly clarify how OPG is involved in vasculogenesis.
228/381
[P142] Contribution of Isotopic Mass Spectrometry to Profiling protein Changes in Colorectal Cancer disease (CRC)
Julien Peltier2, Jean-Pierre Roperch1, Stphane Audebert2, Iradj Sobhani1, Luc Camoin2
1
Service de gastroentrologie & EA4393 laboratoire d'investigation clinique, Universit Paris-Est Crteil, Hpital Henri Mondor, 51, avenue du Mal de Lattre de Tassigny, 94010 Crteil
2
Marseille Protomique, Universit de la Mditerrane, Centre de Recherche en Cancrologie de Marseille INSERM U-891, Institut Paoli-Calmettes, 27, Bd Le Roure, 13273 Marseille Contact: Luc Camoin (luc.camoin@inserm.fr)
Keywords: Clinical proteomics, Proteins, peptides and small molecules quantification Background: The CRC is prevalent, with high cost and bad prognosis. Large polyps in the colon or rectum are considered as precancerous situation in CRC carcinogenesis schema. There is no sensitive and highly specific protein marker available in serum to detect patients with large polyp and CRC. The aim was to identify a deep proteomic library in patients with colon or rectal tumors. Methods: Serum from 4 clinical conditions were obtained: N=normal colonoscopy, A=patients with one polyp of 1 cm or more, K=patients with invasive carcinoma, B=patients investigated after treatment. For each condition, serum was considered for this study (before colonoscopy for N, A, K and 6 months after cancer treatment for B). Samples were submitted to immunopurification (MARS-14 columns), an enzymatic fragmentation followed by a chemical labeling and an isoelectric OffGel separation. Discriminative quantification of proteins was performed according the iTRAQ labeling. Results obtained by mass spectrometry were submitted to ProteinPilotTM (Applied Biosystems) program linked with IPI data bank limited to human proteins. Final analysis were performed by using Cluster 3.0, KEGG Pathway Database and Ingenuity Pathway Analysis (IPA) allowing functional estimation and genes which are involved in the carcinogenesis. Results: After optimization of protein concentration, depletion, trypsinisation and isotopic labeling, a separation of peptides was done based on two dimensional points (isoelectric & hydrophobic). Mass spectrometry MS/MS analysis allowed identification and quantification corresponding to 324 proteins (CI 95%). Based on N as control reference, 100 discriminative proteins were quantified (A/N=29, K/N=59, K/A=55). The proteins belong to two mains biological pathway; inflammation and blood coagulation. Majority of these proteins interact with MMP9 and P53 proteins in particular between N and A. In condition B these two processes are not significantly increased as compared to N. Conclusion: Inflammation and blood coagulation pathways are stimulated soon in the carcinogenesis schema of CRC. Proteins of these two pathways are now under validation in different clinical situations including patients with normal and abnormal colonoscopy. Link: http://map.univmed.fr/
229/381
[P143] The budding yeast proteome revisited: a novel method applied to comprehensive protein identification in a simple eukaryote
Rgis Lavigne1, Emmanuelle Becker 2, Yuchen Liu 3, Bertrand Evrard 3, Michael Primig 3, Charles Pineau1
1 2 3
Proteomics Core Facility Biogenouest, Campus de Beaulieu, 35042 Rennes, cedex, France University Rennes 1, Campus de Beaulieu, 35042 Rennes, cedex, France IRSET, Campus de Beaulieu, 35042 Rennes, cedex, France
Contact: Rgis Lavigne (regis.lavigne@univ-rennes1.fr)
Keywords: Systems biology To reliably identify all proteins present in a given cell or tissue remains a challenge. In the unicellular eukaryote Saccharomyces cerevisiae, much progress has been made by establishing elaborate prefractionation protocols in combination with ever more efficient mass spectrometers. Recently, a method based on SILAC protein labeling has yielded quantitative measurements of protein concentrations in different yeast cultures. In spite of these advances the efficient and cost-effective analysis of whole sets of samples remains extremely laborious and cells cannot be studied under conditions which preclude standard amino-acid labeling. A total yeast protein extract from logarithmically growing diploid cells cultured in rich medium was run on an SDS-polyacrylamide gel, cut into 30 bands and digested with Trypsin. Samples were then processed by iterative nanoflow LC-MS/MS analysis, the generation of peptide exclusion lists to simplify the task of peptide mapping after each round of injection, and an iterative database search with the SEQUEST and Mascot protein identification algorithms. Our label-free method based on a simple protein extraction and separation step identified essentially all known proteins present in vegetatively growing diploid yeast cells. The vast majority of the proteins were found in both replicate experiments. Iterative protein identification was found to substantially increase the number of detected proteins. Simple yeast protein extraction and SDS-gel based prefractionation in combination with our novel mass-spectrometric method yields the proteins present in vegetatively growing yeast cells. This finding raises the exciting possibility of large-scale dynamic proteome profiling of the yeast life cycle in wild type and mutant backgrounds. Moreover, our method is also suitable for studying processes not amenable to SILAC such as meiosis and gametogenesis. Link: http://www.proteome.univ-rennes1.fr/
230/381
[P144] Aspect fondamental de l'utilisation de masques en MALDI

Laurent Diologent1, Maxence Wisztorski1, Grard Bolbach3, Cristian Focsa2, Michael Ziskind2, Isabelle Fournier1
1
Laboratoire de Spectromtrie de Masse et de Biologie Fondamentale & Appliques, EA 4550, Universit Lille 1 Sciences et Technologies, F-59655 Villeneuve d'Ascq
2
Laboratoire de Physique des Laser, Atomes et Molcules, CNRS UMR 8523, Universit Lille 1 Sciences et Technologies, F-59655 Villeneuve dAscq
3
Laboratoire des biomolcules, CNRS UMR 7203, Universit Paris 6, F-75252 Paris
Contact: Laurent Diologent (Petrak@hotmail.fr)
Keywords: Instrumentation, Imaging mass spectrometry Depuis son introduction, fin des annes 80, le MALDI sest impos comme une source de production dions incontournable pour les applications biologiques en permettant danalyser des molcules jusqu des trs haut poids molculaires, quelque soit leur polarit et sous leur forme intacte. Les domaines dapplication du MALDI se sont encore accrus avec le dveloppement de limagerie MALDI la fin des annes 90. Cependant, les mcanismes impliqus dans le processus de dsorption/ionisation sont extrmement complexes et encore mal connus. Ainsi, si la source MALDI permet danalyser de faibles quantits danalytes en partie compatibles avec les systmes biologiques tudis, il faut noter que seul au maximum quelques pourcents dions sont forms par tir laser sur lensemble de la matire jecte. Laugmentation de la quantit dions produite apparat dans ces conditions comme un aspect crucial et particulirement important pour limagerie MALDI, puisque lamlioration de la rsolution spatiale de la technologie pour atteindre une rsolution subcellulaire ncessite une augmentation de la sensibilit. En effet, la quantit dions chute de faon assez drastique pour un faisceau plus focalis. Diffrentes tudes fondamentales peuvent tre conduites pour tudier cet aspect. Dans la prsente tude, nous nous sommes plus particulirement intresss aux effets de lutilisation de masques de silicium sur la quantit dions dtects. Les masques sont des wafers de silicium dans lesquels ont t gravs des ouvertures de dimensions identiques et espaces rgulirement afin de former un quadrillage o chaque ouverture constitue un point danalyse travers laquelle le laser atteint lchantillon. Diffrentes configurations de masques peuvent tre fabriques par nanotechnologies i.e. masques flancs inclins ou flancs droits. Pour une configuration de masque donne, il est galement possible de faire varier les paramtres dimensionnels soit la largeur de louverture et lpaisseur du masque. Les signaux mesurs sur les spectres MALDI-TOF obtenus lors de lutilisation de ces masques se sont montrs grandement influencs par les variations des dimensions de ces masques. Des effets particulirement importants ont t observs sur les masques bords droits. Ainsi, lutilisation de ces masques a clairement montr que les ouvertures les plus petites permettent dobtenir des signaux gnralement plus intenses quen labsence de masque. Ces effets ont t tudis de manire systmatique pour diffrentes conformations afin de comprendre les phnomnes fondamentaux impliqus. Lune des hypothses permettant dexpliquer cette augmentation de signal serait un confinement de la plume lintrieur de louverture amplifiant les collisions efficaces entre les composs dsorbs et augmentant donc le taux dions produits par transfert de proton de la matrice lanalyte. Link: www.maldi-imaging.com
231/381
[P145] MALDI imaging and profiling on a "mirror" mouse model for absence epilepsy: Identification and validation of potential protein markers
Mlanie Lagarrigue1, Theodore Alexandrov2, Rgis Lavigne1, Gabriel Dieuset3, Benot Martin3, Charles Pineau 1
1 2 3
Proteomics Core Facility Biogenouest, IRSET, Campus de Beaulieu, 35042 Rennes, France Center for Industrial Mathematics (ZeTeM), University of Bremen, 28334 Bremen, Germany Inserm U642, Campus de Beaulieu, 35042 Rennes, France
Contact: Charles Pineau (charles.pineau@univ-rennes1.fr)
Keywords: Imaging mass spectrometry, Bioinformatics et biostatistics dedicated to proteomics Childhood Absence Epilepsy is a prototypic form of generalized non convulsive epilepsy characterized by short impairments of consciousness concomitant with synchronous and bilateral spike-and-wave discharges in the electroencephalogram. Two mouse lines BS/Orl (sensitive) and BR/Orl (resistant), constituting a "mirror" mouse model of human absenceepilepsy, were derived from a genetic selection to study absence epilepsies. MALDI imaging mass spectrometry (IMS) was chosen to investigate spatial distribution and expression levels of proteins in brains of the BR/Orl and BS/Orl mouse strains. Frozen brains of four BS/Orl and four BR/Orl were cut on a cryostat and thaw mounted onto Indium-Tin-Oxide coated conductive glass slides. After washing and drying of tissue sections, matrix was applied by vibrational vaporization under controlled conditions using an ImagePrep station. Protein mass spectra were acquired on Autoflex III Smartbeam MALDI-TOF mass spectrometer at 80 m lateral resolution in a mass range of 2-25 kDa. Each dataset constituted of acquired mass spectra during each MALDI imaging experiment was reduced taking only each 16th spectrum (decimation). Null spectra were excluded and mass spectra recalibration was performed. A new type of cross-classification based on a combined discrete wavelet transformation (DWT) - support vector machine (SVM) classification was developed to classify all sets of one type vs. each set of the other type. Very high recognition rates (87-99%) were obtained to classify the BS/Orl and BR/Orl mice brains. Nineteen m/z ratios were thus highlighted as potential biomarkers. Five of them are significant for 7 or 6 out of 8 carried out cross-classifications. Potential biomarkers evidenced by the statistical analysis were further identified using a topdown approach. Proteins from three different zones of mice brains (thalamus, cortex and somatosensory cortex) containing potential biomarkers were extracted and then fractionated by reversed phase HPLC. An aliquot of each collected fraction was analyzed by MALDI-TOF mass spectrometry to select fractions containing proteins with m/z values that could correspond to the biomarkers of interest. Selected fractions were then loaded onto a nanoLC column coupled to an LTQ-Orbitrap XL mass spectrometer for top-down analysis. MS/MS fragmentations were performed using Higher Energy Collision Induced Dissociation (HCD). Several of the most significant potential biomarkers were identified so far, among which a key protein yet suspected to be involved in epilepsy. The topography of expression of candidate proteins was further validated by immunohistochemistry and Western blot experiments. Functional assays are being performed to confirm the involvement of one specific protein candidate in the mechanisms underlying absence epilepsy. Link: http://www.proteome.univ-rennes1.fr/
232/381
[P146] IMPACT OF HUMAN PLASMA PATHOGEN INACTIVATION BY UVIRRADIATION IN PRESENCE OF METHYLENE BLUE/UV light: IDENTIFICATION OF FIBRINOGEN MODIFICATIONS
Alexia ORTIZ1, Christine DEFER1, Fabrice BRAY2, Dominique DERNIS1, Jean-Jacques HUART1, Caroline TOKARSKI2, Christian ROLANDO2
1 2
Etablissement Franais du Sang Nord-de-France, 96, rue Jemmapes, 59012 Lille Cedex
USR CNRS 3290 Miniaturisation pour l'Analyse, la Synthse & la Protomique et IFR 147 Protomique, Modification Post-traductionnelles et Glycobiologie, Universit de Lill 1, Avenue Paul Langevin, 59655 Villeneuve d'Ascq Cedex Contact: Alexia ORTIZ (alexia.ortiz@ed.univ-lille1.fr)
Keywords: High mass and high resolution mass Spectrometry, Clinical proteomics One of the objectives of the French Blood Bank is to preserve the transfusional quality of plasma. Various procedures routinely used for pathogen inactivation cause some loss of biological function in key plasma proteins. The most used inactivation technique is the irradiation of plasma by UV light using methylene blue (MB) as a sensitizer, which is known to affect the biological activity of fibrinogen. Indeed MB, a phenothiazine dye compound, generates reactive oxygen species that oxidize AND, lipids and also proteins in presence of UV light. Fibrinogen is a phophorylated glycoprotein composed of a dimer of three non-identical A-, B-, and -chains that is involved in the clotting process, . This study aims to characterize the impact of BM-treatment on this coagulation factor. First we optimized the isolation of intact fibrinogen. We started the isolation by the classical Cohns fractionation of plasma. Cohns fraction I, which contains fibrinogen, is obtained by cold 8% ethanol at -3C. The fraction was then purified by HIC that does not require the use of organic solvents preserving the protein structure. The stationary phase, which is non commercially available, was obtained by grafting n-pentylamine on Sepharose 4B. A gel filtration step on Superose was added as a polishing step. Fibrinogen activity was checked after each step by testing its clottabilty using thrombin. Plasmas from a single donor treated with MB and native were provided by the the French Blood Bank, Nord de France. Native 1DE clearly highlighted the presence of fibrinogen in its intact form (340 kDa) after the chromatographic steps. Native 1DE also shows the improvement of extra proteins elminination during the purification step, mainly APOB100 by Cohns precipitation and albumine by the HIC column. The identification of the band at 340 kDa was confirmed by in-gel digestion of high molecular weight zones and MALDI-TOF/TOF analysis. Denaturating 1D SDS-PAGE allowed the three , , chains to be separated. 2DE-gel images exhibited a change in the electrophoretic patterns of each subunit. Purified fibrinogen was digested in-solution and the resulting peptides were then analyzed by nanoLC-nanoESI-FT-ICRMS. A label-free quantification pointed out an oxidation in MB-treated fibrinogen on its subunit. A deamidation was also identified on the -subunit of fibrinogen on BM-treated samples. Preparative purifications are now planned to fully establish the link between clottability and these chemical modifications of fibrinogen after UV irradiation in presence of methylene blue.
233/381
[P147] Identification and absolute quantification of a potential degradation marker of transfusion-grade human plasma during production or storage
Alexia ORTIZ1, Latifa RICHA1, Christine DEFER1, Cathy LANE3, Carole MINSINI3, Dominique DERNIS1, JeanJacques HUART1, Caroline TOKARSKI2, Christian ROLANDO2
1 2
Etablissement Franais du Sang Nord-de-France, 96 rue de Jemmapes, 59012 Lille Cedex, France
USR CNRS 3290 Miniaturisation pour l'Analyse, la Synthse & la Protomique et IFR 147 Protomique, Modification Post-traductionnelles et Glycobiologie, Universit de Lille 1, Avenue Paul Langevin, 59655 Villeneuve d'Ascq Cedex, France
3
AppliedBiosystems Sciex, , Warrington, UK
Contact: Alexia ORTIZ (alexia.ortiz@ed.univ-lille1.fr)
Keywords: Proteins, peptides and small molecules quantification, Clinical proteomics The objective of the French Blood Bank is to preserve the transfusion quality of plasma. The plasma retinol binding protein 4 (RBP4) has previously been identified as a potential marker of plasma thermal degradation during production or storage. This 21 kDa protein is responsible of the transport of vitamin A. It circulates associated with transthyretin (TTR) in a tetrameric form to prevent its premature loss by kidney filtration. The aim of the present work is to develop a sensible and reliable method of absolute quantification of free RBP4 and of the RPB4-TTR complex in transfusion plasma. The isolation of both free RPB4 and TTR-complexed RBP4 from plasma sample was set up by coupling anti-RBP4 antibodies which recognize both forms on a Sepharose column. Characterization was performed by 1D gel electrophoresis under both native and denaturing conditions. Bands of interests were digested by trypsin and analyzed by MALDI-TOF/TOF. Then, purifications were achieved according to the French Blood Banks specifications: 6 hours, 12 h, 24 h and 72h after collecting the blood from the donor. To define specific peptides to be labeled by stable isotopes, an in-solution digestion of a commercial RBP4 using trypsin was achieved. The resulting peptides were analyzed by nanoLCnanoESI-Q-TOF MS. Three specific peptides (YWGVASFLQK, FSGTWYAMAK and DPNGLPPEAQK) were identified for the AQUA quantifications and synthesized with isotope incorporation (on 13C and 15N labeled lysine). 5 g of non-depleted plasma were digested in liquid; the resulting peptides were added to the labeled ones in isotopic dilution conditions and analyzed by nanoLC-nanoESI-FT-ICR MS. The total amount of RBP4 was quantified at 1.6 pmol/L. The result is consistent with the theoretical plasma level. Both forms of RBP4 were also quantified after immuno-purification from specifically degraded plasma samples. Additional analyses were achieved on nanoLC-nanoESI-QTRAP MS on non-depleted plasma. RBP4 was selected for MRM assay development from MS/MS data. Linear Ion Trap MS/MS spectra confirmed the identity of the RBP4 peptides. Furthermore, a deamidation was pointed out on the LIVHNGYCDGR peptide. This modification is characteristic of protein degradation. A quantification of this deamidation was achieved from MRM traces. It was also applied to plasma samples degraded under specific conditions related to pathogen inactivation treatments. The time dependence of the apparition of free RBP4 and of post-translational modifications will be discussed.
234/381
[P148] Dtermination par couplage GC/TOF des diffrents composants des huiles essentielles de Zanthoxylum zanthoxylodes et Newbouldia laevis
P. A. Olounlad1, E. Leroy5, E. V. B. Azando1, M. S. Hounzangbe-Adot1, T. B. Tam Ha2, C . Moulis3, N. Fabre3, H. Hoste4, A. Valentin3
1
Laboratoire d'Ethnopharmacologie et de Sant Animale, Facult des Sciences Agronomiques, Universit d'Abomey-Calavi, 01 BP 526, 00229 Cotonou, Bnin
2
Laboratoire de Pharmacognosie de lUniversit Paul Sabatier, 35 Chemin des Marachers, 31062 Toulouse, Cedex France
3
Pharma-Dev -UMR 152 / Facult de Pharmacie, Universit de Toulouse III, 35 Chemin des Marachers, 31062 Toulouse, Cedex France
4 5
UMR 1225 INRA/ ENVT, 23, Chemin des Capelles, 31076 Toulouse Cedex France
Service Commun de Spectromtrie de Masse, Universit Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 09 France Contact: E. Leroy (leroy@chimie.ups-tlse.fr)
Keywords: High mass and high resolution mass Spectrometry, Separative analysis methods Languillulose est une parasitose des zones tropicales et subtropicales dont les traitements perdent de leur efficacit (mergence de rsistances). Au Bnin, la flore qui regorge dune ressource abondante et diversifie de plantes mdicinales et de la connaissance de la mdecine traditionnelle sont susceptibles d'ouvrir de nouvelles voies pour la recherche de nouveaux mdicaments antiparasitaires. Notre travail prsente une dtermination par couplage GC/TOF et valuation in vitro de l'activit anthelminthique des diffrents composants des huiles essentielles de Zanthoxylum zanthoxylodes et Newbouldia laevis, deux plantes mdicinales de la pharmacope traditionnelle bninoise. Environ 81% de composs ont t identifies dans l'huile essentielle des feuilles de Newbouldia laevis alors qu'environ 85% de composs ont t identifies dans l'huile essentielle des graines de Zanthoxylum zanthoxylodes. L'huile essentielle des graines de Zanthoxylum zanthoxylodes prsente 27 molcules dont les principales sont -terpinne (18 %), undcane (14,8 %), valencne (8,3 %), le dcanal (8,3%) et 3 -carne (6,7%). L'huile essentielle des feuilles de Newbouldia laevis contient 42 composs et ses principaux constituants sont -caryophyllne (36,1%), et l'eugnol (5,7%). Les huiles essentielles des deux plantes ont inhib lexcrtion des ufs et la migration des larves de Strongylodes ratti, modle proche de Strongylodes stercoralis agent pathogne de languillulose humaine. Lutilisation traditionnelle de ces plantes dans la mdecine traditionnelle comme anthelminthique semble tre justifie.
235/381
[P149] AT_CHLORO, a Comprehensive Chloroplast Proteome Database with Subplastidial Localization

Myriam Ferro1, Daniel Salvi2, Sabine Brugire1, Daphn Seigneurin-Berny2, Mourad Mellal1, Lucas Moyet2, Christophe Bruley1, Christophe Masselon1, Norbert Rolland2
1 2
BGE/EDyP, CEA/Grenoble, 17 rue des Martyrs, 38054 Grenoble PCV, CEA/Grenoble, 17 rue des Martyrs, 38054 Grenoble
Contact: Myriam Ferro (myriam.ferro@cea.fr)
Keywords: Systems biology Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions. Molecular & Cellular Proteomics 9:10631084, 2010. Link: http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/
236/381
[P150] Gel-free and Gel-based Quantitative Proteomic Profiles of Marine Bacterium Photobacterium angustum Exposed to UVB Radiation
Sabine Matallana-Surget1, Fabien Joux1, Ruddy Wattiez2, Philippe Lebaron1
1
the
Laboratoire dOcanographie Microbienne, Observatoire Ocanologique, Universit Paris VI. CNRS, UMR7621 , Avenue Fontaul, 66650 Banyuls sur mer, France
2
Department of Proteomics and Microbiology, Interdisciplinary Mass Spectrometry Center (CISMa), Universit de Mons, Avenue du Champ de Mars n6, 7000 Mons, Belgium Contact: Sabine Matallana-Surget (sabine.matallana@obs-banyuls.fr)
Keywords: Proteins, peptides and small molecules quantification The marine heterotrophic bacterium with sequenced genome Photobacterium angustum was previously found to be resistant towards UVB radiation. The objective of this study was to examine the UVB response of this bacterial model using both gel-free and gel-based quantitative proteomics approaches. Bacterial cells were grown in artificial sea water with glucose (3 mM) under moderate irradiation (1 W/ m) to study their capacity to photoadapt and to resist to UVB. Irradiated cells and their respective dark controls were harvested for proteomics study after 1h45 of growth under UVB (corresponding to 6.5 kJ / m). The analysis of the changes in the proteome of P. angustum when exposed to UV radiation was analyzed by application of both post-digest Isotope Coded Protein Labeling (ICPL) and 2D DIGE methods. Thus, we used these two quantitative proteomics methods to screen for heart proteins that are regulated under UV radiation. A total of 993 proteins were identified by LC-MS/MS, corresponding to 22% of the theoretical proteome of P. angustum. Four different biological replicates were carried out for each of the two quantitative proteomics methods and a total of 8 LC-MS/MS runs were performed for the shotgun identification. An average of 400 proteins was quantified in all biological replicates of each ICPL run with a differential protein abundance ratio (UVB/DARK) ranging from 0.5 up to 3.5. A total of 583 ICPL labeled non-redundant proteins had significant differential abundance in comparison to the dark control, and 245 were identified in the four replicates, representing an overall average of 25% of the expressed proteome. Among the 245 common proteins in the four biological replicates, 40 were found to be identically regulated. The 2D DIGE technology enabled to identify 22 proteins newly quantified proteins compared to the post-digest ICPL approach, and confirmed the differential regulation of 8 proteins already quantified using the ICPL approach. The major consequence of long term UVB irradiation is DNA damage. Our data are consistent with this, indicating that recombinase A with an average ratio (UVB/dark) of almost 3, play a crucial role in the so-called dark repair systems, involved in excision repair, postreplication recombinational repair, mutagenic or SOS response, and thus preventing the cell against the accumulation of DNA damage. Several other up-regulated proteins are of interest such as a putative antioxidant AhpC/Tsa family protein, chaperonin proteins, and glyoxalase protein as well as a putative lipoprotein. The down-regulated proteins are mainly involved in amino acids transport and metabolism, phosphotransferase system, as well as the Krebs cycle. Currently, we further developed different strategies to confirm and validate those interesting data. Link: http://lomic.obs-banyuls.fr/fr/equipe_biodiversite_et_biotechnologie_microbienne.html
237/381
[P151] Quantitative proteomics ICPL analysis reveals alteration of several functional processes in glioblastoma multiforme
Emmanuelle Com1, Anne Clavreul2, Mlanie Lagarrigue1, Sophie Michalak3, Philippe Menei2, Charles Pineau1
1 2 3
Plate-forme protomique Biogenouest, IRSET , 263 avenue du Gnral Leclerc, 35042 Rennes cedex, France Dpartement de Neurochirurgie, CHU dAngers, Inserm U646, 4 rue Larrey, 49933 Angers, France Dpartement de pathologie cellulaire et tissulaire, CHU dAngers, 4 rue Larrey, 49933 Angers, France
Contact: Emmanuelle Com (emmanuelle.com@univ-rennes1.fr)
Keywords: Clinical proteomics Glioblastoma multiform, the most frequent primary tumor of the central nervous system, remains one of the most lethal human cancers despite intensive researches. The current paradigm in the molecular biology of glioblastoma has been focused on interpatient variability and on trying to isolate new classification elements or prognostic factors. On the contrary, the purpose of the Grand Ouest Glioma Project, a comprehensive pluridisciplinary and multicentric research project financed INCa, is to offer an innovative view on the intra-tumor heterogeneity by analyzing four regions of interest (necrosis, tumor core, interface and peritumoral brain tissues) with different technologies (functional RMI, isolation of tumor cells by primo-cultures from per operative computer guided biopsies, genomics, transcriptomics, proteomics and comparative biological studies). In the proteomics workpackage, we aimed at identifying proteins differentially expressed between the four regions of glioblastoma. It was conducted on biopsies from 5 patients using the ICPL technology. Proteins of the different region of interest were extracted from isolated cells and labeled with light (12C), medium (2H4) or heavy (13C6) ICPL reagents. The three samples were combined and the mixture was further resolved onto a SDS-PAGE gel. The gel lane was cut into 20 pieces that were further trypsin digested. Proteolytic mixtures were analyzed by nanoLC-ESI-ITMS. Protein identifications were obtained by submitting MS/MS data to protein database (SwissProt) search using the Mascot algorithm. Relative quantification was obtained by comparing the signal intensities of light, medium and heavy labeled peptides on MS-spectra, using the WarpLC software. The Annotation, Mapping, Expression and Network (AMEN) software was used to identify the involvement of differential proteins in various biological processes. 584 non-redundant proteins were identified and a protein expression gradient was observed from the tumor core to the periphery. We have identified 31 up-regulated proteins in the tumor region compare to the peri-tumoral brain tissue, among which, 23 proteins belong to an interaction network linked to 4 biological processes such as synaptic transmission. The core of this network is mainly constituted of interactions between beta-actin (ACTB) with heat shock proteins (HSP90AA1, HSPA8) and 14-3-3 proteins (YWHAZ, YWHAG, YWHAB). Furthermore, a cluster of three isoforms of the sodium pump alpha subunit (ATP1A1, ATP1A2, ATP1A3) was identified outside this network. The differential expression observed for ACTB and 14-3-3 gamma was validated by western blot and immunohistochemistry. This study confirms the identity of original molecular targets, highlights several functional processes altered in glioblastoma such as energy metabolism and synaptic transmission and suggests new therapeutic trails.
238/381
[P152] Accessing the testicular germ cell secretome with combinatorial omics: a major breakthrough in deciphering the germ cell-sertoli cell dialogue
Emmanuelle Com1, Frdric Chalmel2, Rgis Lavigne1, Nolwen Hernio1, Laetitia Guillot1, Ana-Paula Teixeira 3, Jean-Louis Dacheux 3, Charles Pineau 1
1 2 3
Plate-forme protomique Biogenouest, 263 avenue du Gnral Leclerc, 35042 Rennes Cedex IRSET, 263 avenue du Gnral Leclerc, 35042 Rennes Cedex UMR INRA-CNRS 6175, Centre de Tours, 37380 Nouzilly
Contact: Emmanuelle Com (emmanuelle.com@univ-rennes1.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics, Systems biology The extremely complex structural organization of the mammalian testis creates particular difficulties for studying its organization, function and regulation. Spermatogenesis that takes place in the seminiferous tubules is an intricate and highly specialized process whose control incorporates juxtacrine, paracrine and endocrine factor information, and is conditioned by the successive activation and/or repression of thousands of genes and proteins, including numerous testis-specific isoforms. It has been yet established that germ cells modulate somatic Sertoli cell function via diffusible proteins. However, the impossibility to maintain germ cells in vitro makes it difficult to study their secretome. Interestingly, within the seminiferous tubules, germ cells and Sertoli cells are surrounded by the testicular fluid, which probably contains factors involved in the germ cell-somatic cell crosstalk. An innovative approach combining testicular fluid collection by microsurgery, fluid prefractionation, shotgun mass spectrometry and combinatorial omics was used to decipher and mine the rat and ram testicular fluids. Over 1400 non redundant proteins were identified and their presence in testicular fluids was further correlated with the transcriptome of isolated testicular cells so as to confirm their cellular origin. Secreted proteins were identified and scored using the Secreted Protein Database (SPD). We demonstrate here that a subset of proteins is actively secreted into the testicular fluid by germ cells. Potential known partners of these germ cell-secreted proteins were proposed using protein network data available via certified public repositories and those known to be expressed on Sertoli cells plasma membranes were selected for further studies. Coexpression of potential germ cell-Sertoli cell protein partners was validated using immunohistochemistry on rat testis sections. Our results provide new insights into the germ cell-Sertoli cell crosstalk by identifying novel interacting protein partners. We also demonstrate that combinatorial omics is a powerful approach to characterize the testicular germ cell secretome that was so far technically inaccessible. Link: http://www.proteome.univ-rennes1.fr/
239/381
[P153] TOF-SIMS imaging study of laminar distribution of cholesterol in Alzheimer disease

Adina N LAZAR1, Nicolas DESBENOIT3, Claudia BICH3, Vanessa PETIT4, Mai PANCHAL1, David TOUBOUL3, Olivier LAPREVOTE5, Alain BRUNELLE3, Charles DUYCKAERTS1
1 2 3
Laboratoire de Neuropathologie Escourolle, Hpital de la Salptrire, 47 Bd de l'Hpital, 75013 Paris, France Centre de recherche de l'ICM, 47 Bd de l'Hpital, 75013 Paris, France
Centre de recherche de Gif, Institut de Chimie des substances naturelles, CNRS, Avenue de la terrasse, 9 Gifsur-Yvette, France
4
LRTS, IRCM, DSV, Commissariat l'nergie atomique CEA, 18 route du panorama, 92265 Fontenay-aux-roses, France
5
Laboratoire de Chimie-Toxicologie Analytique et Cellulaire, Facult des sciences Pharmaceutiques et Biologiques, Universit Paris Descartes, 12 rue de l'cole de mdecine, 75006 Paris, France Contact: Adina N LAZAR (adina_m31@yahoo.com)
Keywords: Imaging mass spectrometry Alzheimers disease (AD) is the most common form of dementia characterized by extracellular deposition of aggregated amyloid beta peptide and intraneuronal accumulation of tau protein. Several evidences support the involvement of cholesterol in the development of AD: the isoform E4 of apolipoprotein E, the main transporter of cholesterol in the brain, is the strongest known risk factor of AD; cholesterol concentration is increased in AD brains (Xiong 2008, Culter 2004, Puglielli, 2003) and cholesterol accumulates in senile plaques (Panchal 2010). In this work we determined if the higher level of cholesterol in the cortex was due to a homogeneous increase or if cholesterol was accumulated in specific layers. By means of TOFSIMS imaging we confirmed that the total amount of cortical cholesterol was significantly higher in AD than in controls. Cholesterol was unevenly distributed in the cortical layers. By correlating the distribution of cholesterol with the probability of location of a given cortical layer, we found that the increase of cholesterol in AD was mainly significant for cortical layer III. This might be related to a higher concentration of senile plaques in this layer. Up to now, TOF-SIMS analysis of the cortex has been performed on rat or mouse tissue (Altellar 2006, McDonnell 2006, Nygren 2005, Sjval 2004, Touboul 2004, 2005). Only two studies report on human brain analysis by SIMS, concerning the imaging and quantification of aluminium in the cortex of nonencephalopathic renal dialysis patients (Candy 1992) or the subcellular distribution of ironferritin-hemosiderin in hippocampus of AD subjects (Quintana 2007).
240/381
[P154] Analyse de composs ultra-volatiles par GC/MS : application ltude des composs de dgradation dune perfluoroctone.
Guillaume Cazals1, Christine Enjalbal2, Franck Guillen3, Abdelkader Dahmani3
1 2
Laboratoire de Mesures Physiques, Universit Montpellier 2, Place E.Bataillon, 34095 Montpellier Cedex 5
Institut des Biomolcules Max Mousseron, Universit Montpellier 2, Place E.Bataillon, 34095 Montpellier Cedex 5
3
Socit S3F chimie, Universit Montpellier 2, Place E.Bataillon, 34095 Montpellier Cedex 5
Contact: Guillaume Cazals (guillaume.cazals@univ-montp2.fr)
Keywords: Separative analysis methods La chromatographie en phase gazeuse (GC) est loutil de choix pour ltude de composs volatile, le couplage de cette technique la spectromtrie de masse permet une identification de composs au sein de mlanges parfois complexes. Cependant dans le cas de ltude de composs ultra-volatiles (points bullition compris entre -10C et -130C), les conditions classiques danalyse et dinjection en solution ne sont plus adaptes. Nous avons dvelopp au laboratoire une technique danalyse de ces composs base sur une injection head-space et une colonne capillaire ayant des proprits spcifiques pour retenir nos composs dintrts. Cette mthode qui se veut efficace et facile mettre en uvre a t applique avec succs ltude des composs issus de la dgradation thermique dune perfluoroctone. Pour cela, la dodcafluoro-2-mthylpentan-3-one (C6F12O, FK-5-1-12) sous forme liquide est soumise une dgradation thermique, le gaz rsultant est ensuite inject sur four de chromatographie gazeuse (Focus GC, Thermo) selon la technique dinjection head-space. La sparation seffectue en utilisant une colonne capillaire Rtx-200 (Restek), greffe avec des groupements trifluoropropylmethyl polysiloxane, assurant ainsi une affinit optimale avec les composs perfluors dintrt. La dtection est assure par spectromtrie de masse simple quadriple en Impact Electronique (DSQII, Thermo) qui sest rvle tre un mode dionisation trs sensible de nos composs. Cette mthode nous a ainsi permis de sparer et didentifier les diffrents composants issus de la dgradation thermique dun compos perfluor. Link: http://www.ibmm.univ-montp1.fr/index.php
241/381
[P155] Development of an absolute and multiplex MS-based quantification method for E. coli carbon central metabolism protein, and application for creating dynamic predictive models.
Mathieu Trauchessec1,2, Michel Jaquinod1, Gwenalle Bestel-Corre2, Christophe Bruley1, Jrme Garin1, Myriam Ferro1
1 2
EDyP/BGE/iRTSV/CEA, 17 rue des martyrs, 38000 Grenoble, France Metabolic Explorer, Biple Clermont LImagne, 63360 Saint-Beauzire, France
Contact: Mathieu Trauchessec (mathieu.trauchessec@cea.fr)
Keywords: Proteins, peptides and small molecules quantification, Systems biology, High mass and high resolution mass Spectrometry METabolic EXplorer is a green chemistry company seeking to design and invent the next generation of daily products by reducing or eliminating the use or generation of hazardous substances. The strategy to develop an economically viable fermentation process was to genetically engineer high-performance production strains. To that aim, Metex use metabolic engineering of E.coli for industrial production. Metabolic network models allow absolute fluxes through larger networks of central carbon metabolism to be determined. However, these stoichiometric based-models only set aside static prediction. To obtain dynamic prediction models, quantitative omics must be integrated into a systems-oriented framework together with enzymology quantitative data. To accurately quantify protein, different MS-based strategies are available, such as AQUA or QconCAT. Among this list, PSAQ strategy (Protein standard Absolute Quantification), based on full length isotope labeled protein as internal standard for mass spectrometry quantification [Brun et al, 2007], is described as the most accurate. To carry out our study, PSAQ strategy coupled to SRM (Selected Reaction Monitoring) analysis has been chosen to accurately and specifically quantify proteins in E.coli lysat. The challenge of the present study is to obtain high-precision absolute quantification of dozen of proteins in a single SRM analysis without any decomplexification. Indeed, dynamic range of E.coli is known to be roughly 10^4 between the less and more abundant proteins. However, in spite of its good specificity, SRM is not sensitive enough to accurately quantify in a so large dynamic range. To bypass heavy pre-fractionation steps, scheduled SRM mode was set up, allowing sensitivity gain, compared to classical SRM mode. For absolute quantitative proteomics we have produced full-length stable 15N isotopally labelled protein. The purification and quality control of protein standards, crucial for goodquality and accurate measurement, were achieved by Amino Acids composition and isotopic distribution using LC/MS on an Orbitrap mass spectrometer. Accurate quantification of dozen of proteins was obtained on a single run, without decomplexification, in a 10^2 dynamic range, scanning hundreds transitions by scheduled SRM. A comparison has been carried out between the conventional SRM and the scheduled SRM modes. In complex matrices, it clearly appeared that scSRM allowed both better chromatogram definition and noise reduction, resulting respectively in a more accurate quantification and sensitivity gain. With this approach, our aim is to scan and accurately quantify approximately 30-40 proteins in a single run, in a larger dynamic range. With a so rapid accurate quantification method, it will become possible to obtain enough quantitative proteomics data of different E.coli strains for providing dynamic prediction models.
242/381
[P156] Comparison between imac and tio2 for on-line enrichment of phosphopeptides: application for quantitative analysis of hepatic biopsies.
Luc Negroni1, Sophie Geiger1, Stephane Claverol3, Marc Bonneu3, Eric Chevet2, Jean Rosenbaum2, Jean-Marie Schmitter1
1 2 3
CBMN, universit de Bordeaux, Univ. Victor Segalen, 146 rue Lo Saignat, BP68, 33076 Bordeaux cedex U1053, Universit de Bordeaux, Univ. Victor Segalen, 146 rue Lo Saignat, 33076 Bordeaux PGF, universit de Bordeaux, Univ. Victor Segalen, 146 rue Lo Saignat, BP68, 33076 Bordeaux cedex
Contact: Luc Negroni (l.negroni@cbmn.u-bordeaux.fr)
Keywords: Signaling, interactomics and post-translational modifications, Separative analysis methods Phosphoproteomics require an enrichment of phosphopeptides that is usually done by affinity chromatography with disposable tips or in-vial. In the present work, we have implemented an on-line process, including a column packed with titanium dioxide (TiO2). Preliminary tests showed unambiguously the better performance of Ti4+ for phosphopeptide enrichment in comparison to IMAC-Fe3+ (POROS 20 MC). Contamination level was estimated using a simple mixture (tryptic digest of casein-albumin) and confirmed the results of Jensen et al.[1]. Different acidic and saline conditions were tested for sample loading. Several elution conditions were compared and confirmed the crucial role of the loading buffer toward the specificity of TiO2 purification. A simple increase of TFA from 0.1 M to 0.65M (5%TFA) dramatically decreases the non-specific binding of acidic peptides (D/E-rich peptides) and the use of 1 M glycolic acid reinforces the specificity of phosphopeptide enrichment. This last eluent was successfully tested with complex samples from plant (root extract) or animal (liver) origin. A protocol that includes iTRAQ labeling and the preliminary results obtained with biopsies from hepatic carcinoma will also be presented. 1. Jensen SS, Larsen MR: Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 2007, 21:3635-3645.
243/381
[P157] Analyse iTRAQ, comparatif RSLC LTQ-Orbitrap-Velos / NanoLC MALDI-TOFTOF 4800

Marjorie Leduc1, Virginie Salnot1, Christian Federici1, Franois Guillonneau1, Cdric Broussard1, Patrick Mayeux1
1
Plateforme de Protomique Paris Descartes (3P5), 22, rue mchain, 75014 Paris
Contact: Marjorie Leduc (marjorie.leduc@inserm.fr)
Keywords: Systems biology La technique iTRAQ (ABSciex) est une mthode de quantification relative base sur lutilisation dtiquettes isobariques. Ces tiquettes propres chaque chantillon sont fixes sur les amines primaires des peptides aprs digestion trypsique et produisent des fragments spcifiques en MSMS (entre 113 et 121Da). Lintensit de ces fragments permet dtablir des ratios de protines. Dans ce poster sont prsentes les optimisations de fragmentation MSMS ralises sur un sytme RSLC LTQ-Orbitrap-Velos (Thermo) pour lanalyse dun protome complexe marqu liTRAQ prfractionn en Off-gel (Agilent). En effet, la fragmentation CID en LTQ ne permet pas de voir le signal des rapporteurs iTRAQ car leur faible masse les rend instables dans lanalyseur. Pour faire de la quantification, il faut utiliser un autre type de fragmentation, lHCD. LHCD peut aussi tre utilis pour obtenir des fragments participant lidentification. Une nergie de collision plus forte peut tre ncessaire pour lobtention dion rapporteur suffisamment intense. Ainsi, 2 approches de fragmentations ont ts compares dabord une association fragmentation CID+HCD puis une association HCD basse et haute nergie de collision. Par ailleurs, la comparaison entre RSLC LTQ-Orbitrap-Velos et NanoLC MALDI-TOF-TOF 4800 (ABSciex) est base sur les rsultats didentification et de quantification obtenu par ProteinPilot 4 (ABSciex). Enfin, lanalyse par spectromtrie de masse est toffe par lajout de donnes bibliographiques grce aux traitements Ingnuity et Pathway Studio des donnes qualitatives et quantitatives. Link: http://3p5.medecine.univ-paris5.fr/
244/381
[P158] Impact mtabolique de lexposition de plusieurs cytochromes P450 diffrents xnobiotiques par chromatographie liquide couple la spectromtrie de masse haute rsolution
Mlaine Courcoul1, Isabelle De Waziers 1, Marie Anne Loriot1, Hlne Gondelle1, Morgane Moulin1, Catherine Marchetti1, Alice Chort1, Sophie Ayciriex2, Olivier Laprevote2, Philippe Beaune1
1
Bases Molculaires de la Rponse aux Xnobiotiques INSERM UMR-S 775, Centre universitaire des Saints Pres, 45 Rue des Saints Pres, 75006 Paris
2
Laboratoire de Chimie-Toxicologie Analytique et Cellulaire, EA 4463, Facult des sciences pharmaceutiques et biologiques, Universit Paris Descartes, 4 Avenue de l'observatoire, 75006 Paris
3 4
Laboratoire de Toxicologie Biologique, Hpital Lariboisire, 2 rue Ambroise Par, 75010 Paris
Laboratoire de Biochimie, Ple de Biologie Produits de Sant, Hpital Europen Georges Pompidou, 20 Rue Leblanc, 75015 Paris Contact: Mlaine Courcoul (melaine.courcoul@parisdescartes.fr)
Keywords: High mass and high resolution mass Spectrometry, Mthodes d'analyse sparative, Organic and inorganic mass spectrometry Les cytochromes P450 (CYPs) sont des enzymes monooxygnases responsables de la monooxygnation de xnobiotiques et de substrats endognes (1). Actuellement, les tudes mtabonomiques sont ralises principalement sur des chantillons in vivo (tissus, bio-fluides) pour des applications diverses telles que le diagnostique et le suivi de maladies, ou pour identifier des biomarqueurs ou des mcanismes responsables de la toxicit de xnobiotiques (2). Nous tudions linfluence de CYPs humains sur le mtabolisme de composs endognes et sur celui de xnobiotiques toxiques. Dans notre approche in vitro, nous utilisons des cellules de type HepG2 (issues dun carcinome hpatique humain (3)) auxquelles nous faisons surexprimer diffrents CYPs. Ces cellules sont ensuite exposes diffrents xnobiotiques. Lutilisation dune telle approche in vitro permet dobserver les perturbations mtaboliques dues la surexpression dun seul CYP et dobtenir un systme biologique simplifi. Les tudes mtabonomiques sur des cultures in vitro sont peu ralises mais restent possibles grce la sensibilit apporte par la spectromtrie de masse. Dans cette tude, nous utilisons la chromatographie liquide (UPLC) couple un spectromtre de masse de type Q-Tof (Synapt G2 HDMS) associs une prparation dchantillons par extraction liquide-liquide afin de mettre en vidence les perturbations mtaboliques. Nos observations portent autant au niveau des petites molcules polaires (acides amins, acides organiques) que des lipides (acides gras, acides biliaires, hormones strodiennes et strols). Une analyse statistique multivarie des donnes LC-MS est ensuite ralise afin didentifier les biomarqueurs spcifiques de la surexpression des CYPs dans les cellules HepG2 ainsi que de lexposition des xnobiotiques. Rfrences: (1) : Guengerich P. Drug Metabolism Review (2004) 36, No. 2 , 159-197 (2) : Masson P, Couto Alves A, Ebbels T, Nicholson J, Want E Analytical Chemistry (2010) 82, 7779-7786 (3) : Aden DP, Fogel A, Plotkin S, Damjanov I, Knowles BB Nature (1979 dec 6) 282, 615-616
245/381
[P159] Signaling pathways activated by oncogenic mutations of the tyrosine phosphatase Shp2: Quantitative phosphoproteomics and functional analysis
Carine Froment1, Armelle Yart2, Karine Trguer2, Mylne Tajan2, Odile Burlet-Schiltz1, Serge Roche3, Bernard Monsarrat1, Patrick Raynal4
1
Proteomics and Mass Spectrometry of Biomolecules, Institut de Pharmacologie et de Biologie Structurale, CNRS, Universit de Toulouse, 205 route de Narbonne, F-31077 Toulouse, France
2 3 4
INSERM UMR 1048, Universit Toulous III, 1 avenue Jean Poulhs, F-31432 Toulouse, France CRBM, CNRS UMR5237 UM1 & 2, 1919 route de Mende, 34000 Montpellier, France EA4568, Universit Toulouse III, CHU Purpan, 31059 Toulouse, France
Contact: Carine Froment (carine.froment@ipbs.fr) Keywords: Signaling, interactomics and post-translational modifications, High mass and high resolution mass Spectrometry, Proteins, peptides and small molecules quantification Mutations of the Shp2 tyrosine phosphatase give rise to leukemia and solid tumours, and to genetic syndromes - Noonan and LEOPARD - with increased cancer susceptibility, so that Shp2 has been recently defined as an authentic oncogene. However, the mechanisms driving its tumourigenic potential are largely unknown. Shp2 is a ubiquitous protein tyrosine phosphatase (PTP) belonging to the Src Homology 2 domain-containing PTPs family. By dephosphorylating specific phosphotyrosine residues, PTPs play essential functions, as they regulate, negatively as well as positively, the signals emanating from tyrosine kinases-dependent membrane receptors (growth factors, cytokines, or hormones receptors, as well as G-protein coupled receptors). Previous reports have suggested that Shp2 could be involved in the Ras/Mitogen-Activated Protein Kinases (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways, possibly through regulation of the Src Family Kinases (SFK). Thus, given the recent identification of these novel Shp2 targets, we aim at deciphering how oncogenic Shp2 mutants affect these signaling pathways and other actors of tumourigenesis. For this study, a series of cellular and molecular tools have been developed to analyse the impact of Shp2 oncogenic mutations notably patient cells (skin fibroblasts, blood cells) and adenovirus/lentivirus infected cells carrying Shp2 mutations. Taking advantage of these tools, we first intend to identify which substrates and more specifically which known or novel phosphotyrosine residues are differentiallydephosphorylated by oncogenic Shp2. To this aim, we chose to use a SILAC quantitative phosphoproteomic approach combining a 13C-tyrosine metabolic labeling and tyrosine phosphorylated peptides enrichment with phosphotyrosinespecific antibody. Briefly, two populations of cell lines (patient versus healthy cells or adenovirus/lentivirus infected cells) were switched from growing in media containing normal tyrosine to media containing 13C9-tyrosine and both lysates were combined prior to trypsin digestion. Then, the peptides containing phosphotyrosine were isolated directly using the PhosphoscanTM Kit (Cell signaling Technology), identified by nanoLC-MS/MS on a LTQ Orbitrap Velos mass spectrometer using CID and HCD fragmentation modes and quantified using the inhouse developed MFPaQ software. Preliminary analyses of cells expressing or not Shp2 and stimulated by EGF allowed to identify and quantify about 300 phosphotyrosine peptides. Among them, 49 phosphotyrosine peptides were differentially phosphorylated. Importantly, almost all these tyrosine phosphorylated peptides belong to proteins involved in proliferation-promoting signaling pathway including potential Shp2 substrates or Shp2 downstream effectors.
246/381
[P160] Dveloppement compar de stratgies analytiques bases sur les couplages GC-MS/MS et LC-MS/MS pour la mesure du Bisphnol A dans les matrices alimentaires.
Yoann DECEUNINCK1, Zita ZENDONG1, Emmanuelle BICHON1, Jean-Philippe ANTIGNAC1, Bruno LE BIZEC1
1 2 3
LABERCA - ONIRIS, Route de Gachet, 44307 NANTES Universit Nantes Angers Le Mans, , INRA, , Nantes
Contact: Yoann DECEUNINCK (yoann.deceuninck@oniris-nantes.fr)
Keywords: Proteins, peptides and small molecules quantification , Separative analysis methods Le bisphnol A (BPA, 4,4-dihydroxy-2,2-diphnylpropane) appartient la famille des diphnylalcanes hydroxyls ou bisphnols. Le BPA est notamment utilis comme monomre pour la fabrication industrielle par polymrisation de plastiques de type polycarbonate et de rsine poxy. Largement prsent dans notre environnement quotidien, ce compos fait lobjet dune attention particulire et trs actuelle de la part la fois de la communaut scientifique et des agences en charge de lvaluation du risque quand ses effets sur la sant de lHomme. Un manque de donnes est en particulier constat quand lexposition et limprgnation des populations ce compos. Dans ce contexte gnral, la spectromtrie de masse apparait comme la technique analytique de choix pour lidentification et la quantification du BPA dans les diverses denres alimentaires. Le mode dacquisition SRM (Selected Reaction Monitoring) est quant lui le plus adapt une mesure la fois sensible et spcifique. Le comportement du BPA en termes de sparation chromatographique, dionisation, puis de schma de fragmentation en MS/MS, a t tudi sur deux systmes de type GC-(EI)-MS/MS et UHPLC-(ESI)-MS/MS. Les signaux diagnostiques (transitions SRM) spcifiques du BPA ont ainsi t dtermins pour les deux approches, et les conditions instrumentales (paramtres dionisation dans la source et de fragmentation dans la cellule de collision) ont t optimises. La quantification du BPA a t assure par dilution isotopique en utilisant le 13C-BPA comme talon interne. Les deux stratgies ont enfin t compares sur la base des limites de dtection observes et du temps danalyse. Paralllement, un protocole de prparation des chantillons a t dvelopp pour lextraction et la purification du BPA partir de diverses matrices alimentaires. Le couplage GC-MS/MS est finalement apparu comme la technique la plus sensible permettant ainsi une identification du BPA dans les diffrentes matrices alimentaires, une concentration infrieure 0,01 g/kg. Dautre part, les rsultats obtenus lors de la phase de validation de la mthode, en termes de reproductibilit et de justesse des rsultats ainsi que la matrise de la contamination environnementale (matriaux et matriels) se sont avrs trs satisfaisants et en adquation avec les niveaux doccurrence publis dans la littrature, i.e. une limite de quantification infrieure 0,1 g/kg. Cette mthode a enfin t valide conformment aux rfrentiels en vigueur et saccompagne dun suivi continu des performances par lintermdiaire de cartes de contrle (matrise des paramtres de contamination analytique et de justesse). Applicable en ltat aux matrices alimentaires et donc la gnration de donnes dexposition, cette stratgie est en voie dtre adapte pour la mesure du BPA dans les fluides biologiques en vue de la gnration de donnes dimprgnation.
247/381
[P161] Caractrisation des glycosylations de linvertase vacuolaire de raisin par combinaison de diffrentes stratgies de spectromtrie de masse
Agns Hovasse1, Sandrine Jgou2, Tchilabalo Alayi1, Alexandra Conreux2, Sandra Villaume2, Alain Van Dorsselaer 1, Christine Schaeffer-Reiss1
1
Laboratoire de Spectrometrie de Masse Bio-Organique, DSA-IPHC, UMR 7178, 25 rue Becquerel, 67087 Strasbourg
2
Laboratoire dOenologie et Chimie Applique, URVVC-SE UPRES EA 2069, Universit de Reims ChampagneArdenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2 Contact: Agns Hovasse (ahovasse@unistra.fr)
Keywords: Signaling, interactomics and post-translational modifications Linvertase vacuolaire de raisin est une enzyme cl du mtabolisme des sucres du raisin implique dans lhydrolyse du saccharose en glucose et fructose et est une des protines majeures des vins de Champagne [1-2]. Cette glycoprotine possde 12 squences consensus de N-glycosylation dont seulement 2 sont actuellement dcrites [3]. La caractrisation complte des glycosylations de linvertase de raisin permettrait notamment de mieux comprendre leur impact sur sa stabilit, sa solubilit, son repliement, son activit biologique, sa rsistance potentielle aux protases et son rle dans les proprits moussantes des vins effervescents dont le Champagne. Nous avons donc ralis une analyse structurale fine des sites de glycosylation et de leur microhtrognit en utilisant une combinaison de stratgies bases sur la spectromtrie de masse. Dans un premier temps, pour dterminer les sites de glycosylation, la protine entire purifie [4] a t dglycosyle par la PNGase A, puis digre (trypsine et chymotrypsine en parallle). Une dglycosylation a aussi t ralise sur la protine pralablement digre la trypsine. Lanalyse de ces 3 digests par nanoLC-MS/MS a mis en vidence la prsence de 9 Asn damides en Asp en positions 108, 225, 277, 289, 430, 450, 499, 621 et 626. Dans un deuxime temps, pour caractriser la micro-htrognit de ces sites, linvertase a t digre par 3 enzymes (trypsine, chymotrypsine et GluC en parallle), et les digests enrichis en glycopeptides (phase ZIC-HILIC). Une premire analyse des 3 digests par nanoLC-MS (Q-TOF maXis), avec recherche des ions dits diagnostiques issus de la fragmentation spcifique des glycanes, a permis de localiser dans les chromatogrammes les glycopeptides et de donner une image de leur micro-htrognit. Une deuxime analyse par nanoLC-MS/MS a permis didentifier les glycoformes prsentes sur 5 sites de glycosylation, de dterminer la masse de la partie peptidique correspondante pour chacun deux et donc de les identifier. Cette stratgie a permis de caractriser la micro-htrognit (jusqu 11 glycoformes) pour 5 sites de glycosylation en positions 158, 212, 430, 450, 485. Les glycosylations observes sont de type complexe ; la majorit porte un fucose en alpha 1-3 sur le GlcNAc proximal ainsi quun xylose. En conclusion, nous avons identifi la totalit des 12 sites potentiels de linvertase vacuolaire de raisin, et caractris la micro-htrognit de 5 de ces sites. Ce fort taux de glycosylation permettra de mieux comprendre limpact des glycosylations de linvertase sur les proprits moussantes des vins effervescents, et sur les caractristiques biochimiques de cette protine. 1. Cilindre C., et al., J Proteome Res 2008, 7. 1199-208 2. Dambrouck T., et al., J Agric Food Chem 2005, 53. 8782-9 3. Palmisano G., et al., J Proteome Res 2010, 9. 6148-59 4. Jgou S., et al., Anal Chim Acta 2009, 638. 75-8 Mots cls : Signalisation, complexes et modifications post-traductionnelles
248/381
[P162] Quantification of homologous proteins in yeast protein fractions that modulate the assembly of the yeast prion Sup35p
Virginie Redeker1, Chris Hughes2, Jimmy Savistchenko1, Johannes P.C. Vissers2, Ronald Melki1
1
Laboratoire dEnzymologie et Biochimie Structurales, CNRS, 1 avenue de la terrasse, 91190 Gif-sur-Yvette, France
2
Waters Corporation, Atlas Park, Simonsway, M22 5PP Manchester, UK
Contact: Virginie Redeker (redeker@lebs.cnrs-gif.fr)
Keywords: Proteins, peptides and small molecules quantification The aggregation of the bakers yeast prion Sup35p is at the origin of the transmissible [PSI+] trait. We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI+] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI+] and [psi-] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes. We and others have shown previously that molecular chaperones modulate Sup35p aggregation (1). Interestingly, in this study, we identify and quantify nineteen proteins involved in protein folding. Among the identified proteins, we find a number of homologous proteins. In order to quantitatively assess individual protein paralogs, we processed manually the data. When isoform specific peptide sequences were detected a quantification of protein isoforms was carried out. Otherwise, the different isoforms were grouped in homology protein group and an absolute amount assigned to the group as a whole. The isoform processing methodology used to quantify a number of protein homologs, and in particular some chaperone protein isoforms, will be presented. Our functional proteomic study provides the first inventory list of over 300 proteins, including 19 chaperon proteins, that directly or indirectly affect Sup35p aggregation and [PSI+] formation. Our results pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified in this study, susceptible of affecting Sup35p assembly. (1) Krzewska J, Melki R., EMBO J. 2006, 25: 822-833
249/381
[P163] Microenvironnement et cancer du sein : analyse protomique du scrtome dadipocytes cocultivs en prsence de cellules tumorales
Karima Chaoui1, Victor Laurent1, Batrice Dirat1, Philippe Valet1, Catherine Muller1, Bernard Monsarrat1, Odile Burlet-Schiltz1
1
Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 route de Narbonne, 31400 Toulouse Cedex, France
2
Institut des Maladies Mtaboliques et Cardiovasculaires, Inserm, CHU Rangueil, BP 84225, Avenue Jean Poulhs , 31062 Toulouse cedex 4, France Contact: Karima Chaoui (Karima.Chaoui@ipbs.fr)
Keywords: Proteins, peptides and small molecules quantification , Systems biology Les adipocytes demeurent peu tudis en dpit du fait quils reprsentent le type cellulaire prdominant du microenvironnement du cancer du sein. Pourtant, des tudes pidmiologiques ont montr que lobsit dans le cancer du sein pouvait tre associe un mauvais pronostic avec des tumeurs plus agressives dfinies par un stade avanc et une propension plus leve donner des mtastases. Des rsultats rcemment publis ont montr que la coculture (2D) de cellules tumorales avec des adipocytes contribue augmenter les capacits invasives des cellules tumorales in vitro et in vivo. Ces rsultats laissent penser quun cross-talk existe entre les deux populations cellulaires et que la population des adipocytes participe la progression tumorale (1). Lobjectif de cette tude a donc t didentifier les protines adipocytaires dont lexpression est module par la coculture avec des cellules tumorales du sein afin de comprendre les mcanismes mis en jeu. Dans ce but, nous avons ralis une analyse protomique quantitative diffrentielle de type label free (2) des scrtomes dadipocytes F442A (dorigine murine) cocultivs durant 3 jours avec des cellules ZR (cellules tumorales du sein humaines) versus un pool de scrtomes dadipocytes et de cellules ZR non cocultivs. Ces deux scrtomes ont t fractionns sur un gel SDS-PAGE et chacune des deux pistes a t dcoupe de faon systmatique. Les bandes obtenues ont t analyses sur un appareil de type LTQ-Orbitrap XL, dont la haute rsolution et la vitesse de squenage permettent lanalyse optimale des mlanges complexes. Cette analyse diffrentielle a t ralise sur trois rplicats dchantillons biologiques et nous a permis de quantifier plus de 2700 protines et dobserver plus de 200 protines variantes entre les deux scrtomes. A titre dexemple, ces analyses ont ainsi permis didentifier diverses chimiokines dont lexpression est fortement diminue par la coculture avec les cellules tumorales. Les chimiokines sont des protines capables de stimuler la migration de certaines cellules tumorales via la formation dun gradient de chimiokine ; la migration se faisant de faible vers de fortes concentrations en chimiokines. Cette rgulation ngative des chimiokines adipocytaires en coculture pourrait correspondre une diminution de lexpression au niveau du front invasif, favorisant ainsi la formation du gradient et donc la migration. Enfin il est noter que la diminution dexpression de ces chimiokines en coculture a t valide par la suite par PCR quantitative. (1) Dirat B et al, Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion, Cancer Res, 2011, 71(7):2455-65. (2) Mouton-Barbosa E et al, In-depth exploration of cerebrospinal fluid by combining peptide ligand library treatment and label-free protein quantification, MCP, 2010, 9(5):1006-21.
250/381
[P164] The secretome of Botrytis cinerea : adaptation to environmental pH

Cindy Dieryckx1, Vincent Girard1, Semcheddine Cherrad1, Dominique Job1, Claudette Job1, Christine Rascle 1, Nathalie Poussereau 1
1
Joint laboratory, University of Lyon1- CNRS UMR5240-Bayer S.A.S., 14-20 rue Pierre Baizet, 69009 lyon
Contact: Vincent Girard (vincent.girard@bayer.com)
Keywords: Separative analysis methods Micro-organisms must adapt to environmental change to survive, and this is particularly true for fungal pathogens such as Botrytis cinerea. B. cinerea is found both in the environment and in diverse plant hosts. Their ambient pHs varie considerably, and therefore we have examined the response of B. cinerea to changes in ambient pH using a proteomic approach. The expression of secreted proteins induced by controlled growth conditions, i.e. complete synthetic medium at pH 5.0, was identified by two-dimensional gel electrophoresis and image analysis software. All reproducible and statistically significant spots were identified by peptide mass fingerprinting, thereby extending our 2-DE map of the Botrytis cinerea to a total of 257 identified proteins. Proteins expressed in B. cinerea cells growing at pH 5.0 or 7.0 were compared by 2-DE. Qualitative and quantitative differences were observed related to the protein patterns whereas at both pH (5.0 and 7.0). Furthermore, characterization of a strain of B. cinerea deleted transcription factor Pacc will clarify the role of the signaling pathway Pal/Pac on the establishment of the secretome of the fungus in both pH. Discussion will be done on the possible factors regulating secreted protein expression by the pH in Botrytis. In addition, our data suggested that different fungal proteins could be involved in the infection process depending on the external pH.
251/381
[P165] ISO 9001, un outil adapt et accessible pour lorganisation et la gestion des plateformes et des quipes scientifiques associes
Renaud Albigot1, Stphane Libat1, Carole Pichereaux1, Marie-Pierre Dubrulle2, Odile Schiltz1, Bernard Monsarrat1
1
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, Universit de Toulouse, 205 Route de Narbonne, 31077 Toulouse, France
2
GIS IBiSA, 147 rue de l'Universit, 75338 Paris, France
Contact: Renaud Albigot (renaud.albigot@ipbs.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics Quil sagisse des plateformes ou des quipes de recherche, le besoin de rpondre dans des dlais contrls aux demandes de prestation ou de collaboration scientifique, la ncessit de consolider la qualit des expertises ralises grce la gestion des comptences du personnel ainsi que la matrise de lensemble du parc instrumental, laisse clairement apparatre la ncessit dencadrer lactivit par la mise en place dune dmarche qualit permettant de rpondre ces besoins dune manire parfaitement structure. Cela rpond galement des enjeux : - Scientifiques : une organisation optimise permet de matriser les cots, la gestion des ressources humaines, mais aussi la fiabilit et la diffusion de linformation (rsultats exprimentaux, publications, brevets) ; - Financiers : limplication dans une dmarche qualit favorise lallocation de fonds car elle garantit les meilleures conditions de production scientifique et la capacit grer les ressources alloues. En nous appuyant sur lexemple dun systme qualit mis en place au sein de lInfrastructure Protomique de Toulouse depuis 2006, nous illustrons limpact du rfrentiel ISO 9001. Impact pour la direction, dans sa gestion quotidienne de ces structures, impact pour le personnel dans son activit journalire et enfin, impact pour les collaborateurs et les clients dans lutilisation de nos infrastructures et de nos rsultats. Ces lments laissent apparatre que le rfrentiel ISO 9001, loin de son image d usine gaz paperassire , savre un outil dorganisation dune grande souplesse, dune mise en uvre relativement simple et largement compatible avec les spcificits des plateformes technologiques et des quipes de recherche. Link: http://proteomique.ipbs.fr
252/381
[P166] Diurnal metabolite changes of red ripe tomato fruit and mature leaf using mass spectrometry methodologies
Stphane Bernillon1, Camille Bnard2, Mickal Maucourt3, Benoit Biais1, Sonia Osorio-Algar, Alisdair Fernie5, Emilie Labadie-Lemire1, Patricia Ballias, Catherine Deborde, Annick Moing 1, Ccile Cabasson, Dominique Rolin3, Michel Gnard, Hlne Gautier2, Yves Gibon1
1
INRA, UMR1332, Biologie du Fruit et Pathologie, 71 Avenue Edouard Bourlaux, 33140 Villenave d'Ornon, France
2
INRA UR1115 Plantes et Systmes de culture Horticoles, Domaine St Paul, Site Agroparc, 84914 Avignon, France
3
Universit de Bordeaux, UMR 1332, Biologie du Fruit et Pathologie, 71 Avenue Edouard Bourlaux, 33140 Villenave d'Ornon, France
4
Plateforme Mtabolome du Centre de Gnomique Fonctionnelle Bordeaux, IBVM, Centre INRA Bordeaux, 71 av Edouard Bourlaux, 33140 Villenave d'Ornon, France
5
Max-Planck-Institut of Molecular Plant Physiology, Am Mhlenberg 1, 14476 Golm-Potsdam, Germany
Contact: Stphane Bernillon (stephane.bernillon@bordeaux.inra.fr)
Keywords: Systems biology The Fruit Integrative Modelling project is an Eranet EraSysBio+ project, which aims at describing and modelling the influence of environmental and metabolic variables on the quality of tomato fruits (Solanum lycopersicum cv Money Maker). The present study focused on the importance of diurnal changes in the metabolome of tomato fruits and leaves. For this, red ripe fruits and their closest leaf were harvested at the end of the night, at midday and at the end of the day and analyses of pericarp and foliar limb tissues were performed on water/methanol/chloroform extracts using GC/EI-ToF-MS after derivatization, and on methanol/water extracts using LC/ESIQToF-MS. Identity of metabolites was assigned by comparison with a database for GC/MS data, and by accurate masses, diagnostic fragments and literature for LC/MS data. Metabolite levels were estimated and normalised relatively to a biological standard. A multivariate analysis revealed that the metabolome of red ripe fruits undergoes subtle changes throughout a day and night cycle. Several metabolites were then found to change significantly during the day, but with very low amplitude. In contrast and as expected, metabolite fluctuations were of much larger amplitude in leaves. The next step of this study will be to investigate the metabolism of the fruit at green stages throughout the same diurnal cycle.
253/381
[P167] Proteomic analysis of S-nitrosylation induced by the lipid A analogue, OM174

Myriam Lamrani1, Graldine Lucchi2, Patrick Ducoroy2, Jean-Franois Jeannin1, Ali Bettaieb1
1
Laboratoire dImmunologie et Immunothrapie des cancers INSERM U866 - Facult de Mdecine, 7 Boulevard Jeanne dArc, 21000 Dijon
2
CLIPP IFR100 CHU Universit de Bourgogne, 1 rue du Professeur Marion, 21000 Dijon
Contact: Patrick Ducoroy (Patrick.Ducoroy@clipproteomic.fr)
Keywords: Signaling, interactomics and post-translational modifications Background: The antitumor efficacy of a lipid A analogue (OM-174) was demonstrated, an effect that requires the expression of NOS II and the production of nitric oxide (NO) by tumor cells. Recent studies show that NO can react with cysteine residues target proteins to form Snitrosothiols (SNOS), a process called S-nitrosylation, thereby altering protein function (inhibition, activation). Methods: To characterize the profil of S-nitrosylated proteins, murine mammary cancer cells EMT-6H were treated or not by OM-174 combined with IFN gamma (a cytotoxic combination). S-nitrosylated proteins were detected by the method of Biotin Switch Assay (BSA). This method involves replacing the S-NO bond cysteines of target proteins by S-biotin bonds, followed by purification on a streptavidin column. The biotinylated proteins (S-nitrosylated) are then reduced, alkylated and digested by trypsin. After purification and concentration of generated peptides for each sample, they are separated by nanochromatography column liquid C18. The 140 fractions generated are analyzed by MALDI mass spectrometry in MS and MS/MS. Results: Sixteen specific proteins in the EMT-6H stimulated by OM-174/IFN gamma combination sample are identified. We are interested in three proteins: both involved in cell survival (nucleophosmin and ubiquitin conjugating enzyme UBC13) and one involved in the anti-tumor immune system activation (ERp57). We confirmed their S-nitrosylation by BSA and a Western Blot. Conclusion: The role of S-nitrosylation in the anti-tumor efficacy of OM-174 on EMT-6H cells remains to be evaluated. The cDNA construct encoding mutated forms of the proteins of interest, on their S-nitrosylated cysteines, will determine the role of these changes. This work should allow the identification of new proteins targets of NO. Their S-nitrosylation may affect their functions leading to lipid A antitumor effect of breast cancer cells. Link: www.clipproteomic.fr
254/381
[P168] Nanostructured materials for the phosphoproteome profiling with mass spectrometry
Graldine Lucchi1, Nicolas Martin2, Joseph Gavoille2, Patrick Ducoroy1, Wilfrid Boireau2
1 2
CLIPP CHU IFR100 Universit de Bourgogne, 1 rue du Professeur Marion, 21000 Dijon FEMTO-ST MN2S Universit de Franche Comt, 32 rue de l'Observatoire, 25000 Besanon
Contact: Wilfrid Boireau (wboireau@femto-st.fr)
Keywords: Signaling, interactomics and post-translational modifications Background: Phosphorylation plays a pivotal role in the regulation of many biological processes and its dysregulation has been found to be the underlying basis of many human diseases[1]. Moreover, phosphoproteins could be cancer markers useful for diagnostics and cancers fingerprinting [2,3]. Mass spectrometry (MS) is the method of choice for the analysis of protein phosphorylation because of its sensitivity, speed, and simplicity [3,4]. However, MS analysis of phosphorylation on a proteome-wide scale remains a major challenge due to many limitations (low abundance & stoichiometry and high dynamic nature of protein phosphorylations) [4, 5]. Methods: Procedures of enrichment of phosphopeptides prior MS analysis are crucial and could be based on metal oxide affinity chromatography (MOAC)[5]. On the other side, during the last decade, the notion of Surface-Assisted Laser Desorption/Ionization (SALDI-MS) appears because the surface structure was critical to obtain mass spectra without chemical matrixes. Research studies on TiO2 based materials are very active and a recent publication has pointed out the potential of Titania substrates in this field [6]. Results: Here, we present the influence of nanostructuration of titania substrates on the phosphopeptides enrichments and SALDI-MS analysis. The optimization of the fabrication of nanostructured titania substrates and the establishment of protocols to promote the on-chip selection of phosphopeptides have allow to detect 3 multi and 4 monophosphopeptides after deposition and treatment of 25 fmoles of alpha-casein digest. Moreover, we have demonstrated the ability of rutile structure to ionize and desorb peptides in SALDI experiments. By this way, BSA has been identified in MS and MS2 modes at fmole level. Conclusion: For the first time, we demonstrated the powerful SALDI and chromatographic properties of rutile among titania substrates for phosphoproteomic studies. References: 1) Nita-Lazar et al.. Proteomics 2008, 8, 443343 2) Lim YP.; et al., Clin Cancer Research 2004; 10, 3980-7 3) Thingholm T. E, et al., Proteomics 2009, 9, 1451 68 4) Steen J. A. J.; et al., Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 606974 5) Bodenmiller B.; et al., Nat. Methods 2007, 4, 231237 6) Bi H. Y., et al. Anal Chem 2009, 81, 1177-83 Link: www.clipproteomic.fr
255/381
[P169] Discrimination between Arabica and Robusta coffee species based on their peptide/protein profiles using ClinProTools approach
Laure F. Marvin-Guy1, Justine Yerly1, Philippe Montavon1, Helia Dias Abelairas1, Dominique Piguet1, Young Joo Chung1, Irma Silva Zollezzi1
1
Nestl Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland
Contact: Laure F. Marvin-Guy (laure.marvin-guy@rdls.nestle.com)
Keywords: Proteins, peptides and small molecules quantification Coffee is the most traded food commodity. The two commercially relevant coffee species are Arabica and Robusta. In the cup, they strongly differ by their sensory profiles, Arabicas providing the highest quality blends and being more expensive than Robustas. Hence, tools to evaluate the purity of Arabica blends are of commercial importance. Furthermore, green and roasted coffee blends appear on the market. The identification of coffee proteins in these mixtures might be required to solve quality issues. Here we show that coffee protein extracts, produced from commercial Arabica and Robusta green coffee, can be clearly ascribed to their original source by using Matrix-Assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and data analysis with ClinProTools. This commercially available software allows the visualization of peptide/protein profiles and comparison of datasets with powerful bioinformatic tools. Multivariate analyses applied to our MALDI-TOF-MS data demonstrated strong discrimination power between the two coffee species, which should lead to the discovery of valuable biological markers to evaluate purity and origin of coffee blends. This technique is less time-consuming, more specific and sensitive, and cheaper than other proteomic techniques like e.g. gel electrophoresis.
256/381
[P170] Etude de complexes Cyclodextrine-Gadolinium par ESI-MS

Marie Hubert-Roux1, Ibrahim Zgani1, Hussein Idriss1, Franois Estour1, Graldine Gouhier1, Catherine Lange1
1
CNRS UMR 6014, Universit de Rouen, Rue tesnires, 76821 Mont-Saint-Aignan, France
Contact: Marie Hubert-Roux (marie.hubert@univ-rouen.fr)
Keywords: Organic and inorganic mass spectrometry Dans le cadre de la recherche actuelle en IRM, et plus spcifiquement de la mise au point dune imagerie lchelle molculaire, de nouveaux agents de contrastes (AC) dits intelligents (ou sondes bimodales) sont dvelopps pour permettre de dtecter et de quantifier in vivo les phnomnes biologiques extra-cellulaires. Lagent de contraste choisi est le Gadolinium III (Gd III) li une -Cyclodextrine (CD). En effet, il avait t montr que les CD taient des agents prometteurs pour la vectorisation in vivo des complexes avec le Gd, permettant daugmenter la valeur de la relaxation des protons de leau et par consquent lactivit en terme dimagerie mdicale [1,2,3]. La premire tape de synthse de ces nouvelles sondes a consist greffer sur la Cyclodextrine 7 ligands actate pouvant coordiner le Gd. Ce complexe CD/Gd a t caractris par Spectromtrie de Masse sous ionisation Electrospray (ESI) en mode de dtection ngatif. Certains paramtres source de tension, temprature et pression ont t ajusts afin doptimiser le signal des espces multicharges du complexe. Puis, des chantillons de rapports molaires CD/Gd diffrents ont t analyss par cette technique afin de dterminer la stoechiomtrie des complexes forms. [1] S. Aime, M. Botta, F. Fedeli, E. Gianolio, E. Terreno, P. Anelli, Chem. Eur. J., 2001, 7, 5261-69. [2] S. Aime, E. Gianolio, E. Terreno, I. Menegotto, C. Braco, L. Milon, G. Cravotto, Magn. Res. Chem., 2003, 41, 800-805. [3] S. Aime, E. Gianolio, E. Terreno, F. Uggeri, S. Tagliapietra, A. Barge, G. Cravotto, J. Inorg. Biochem., 2006, 100, 931-938.
257/381
[P171] Identification of the species of animal glues found in artwork by proteomics

Sophie Dallongeville1, Olga Plechakova1, Nicolas Garnier2, Genevive Reille-Taillefert3, Caroline Tokarski1, Christian Rolando1
1
USR CNRS 3290 Miniaturisation pour l'Analyse, la Synthse & la Protomique (MSAP), Universit Lille 1, Cit Scientifique Bat C4, 59655 Villeneuve d'ascq cedex
2 3
SARL Laboratoire Nicolas Garnier, , 63270 Vic Le Comte A.R.T SA (Atelier de Restauration Taillefert) , 37, rue de Gruenewald, Luxembourg
Contact: Caroline Tokarski (Caroline.Tokarski@univ-lille1.fr)
Keywords: High mass and high resolution mass Spectrometry, Bioinformatics et biostatistics dedicated to proteomics Over the past centuries, artists have tried out various organic compounds as binding media to develop their paint recipes. Among these substances, animal glues have been used not only as binders for pigments but also in mixtures with chalk for the preparation of ground in canvas paintings, as adhesives or for gilding. Traditionally animal glues are made by boiling skin or bones of mammals or by boiling sturgeon bladders for fish glue. Different methods have been proposed to identify proteinaceous binders and discrimination between animal glues and egg or milk is easily achieved. Nevertheless, the identification of the origin of the animal glue is still a challenging task. In this study, we propose to identify proteins in animal glue samples and to achieve animal species differentiation on the basis of species-specific peptides. The methodology, based on an adapted proteomics approach including high resolution analysis using nanoLC-nanoESI-Qh-FT-ICR MS/MS was first evaluated on commercially available samples of animal glues from different origins in order to create a database of peptides specific of the different species of interest (i.e. bovine, rabbit and fish). In order to identify peptides specific of a species, homemade software based on Biopython was developed. The specific peptides identified were checked by performing a blast within the ncbi database. As result, up to 15 specific peptides were identified for the bovine glue, including 2 different collagen proteins. Concerning rabbit skin glue, 3 specific peptides corresponding to one rabbit collagen protein were found and in the case of sturgeon glue, 3 fish-specific peptides were identified by sequence homology with the rainbow trout. The methodology was then applied to authenticate different rabbit skin glue samples, including a 60 years old sample coming from Maison Totin-Frres, one of the most famous French manufacturers of rabbit skin glue. The analysis was performed on hundreds micrograms of painting and, for example, 8 bovine-specific peptides were found for the model painting, made according to old recipes, during this work with cow hide glue as binder. Finally, the methodology was applied on a gilt sample coming from the St Maximin church, located in Thionville. Bovine collagens 1 and 2 type I, bovine collagen 1 type II and bovine collagen 1 type III were identified. More interestingly, 4 specific peptides of bovine collagen 1 type I were found. For example, the sequence GEPGPAGLPGPPGER, specific to bovine collagen 1 type I (472-486) was identified by MS/MS using the complete set of y1+ to y13+ ions. 8 specific peptides were identified for collagen 2 type I, including for example the sequence IGQPGAVGPAGIR (1066-1078) and only one specific peptide for collagen 1 type III. These results prove that animal glue from bovine origin was used for the making of this gilt.
258/381
[P172] Caractrisation de la machinerie multiprotique implique dans lArchitecture de lADN Transformasome chez S. pneumoniae : mise en uvre dapproches conjugues de type Tap-Tag et de Protomique Quantitative en Label Free.
Audrey Olivier2, Alexandre Stella1, Jean-Pierre Claverys2, Patrice Polard2, Odile Burlet-Schiltz1, Bernard Monsarrat1
1
Plateforme Protomique Toulouse Midi-Pyrnes, Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, Universit de Toulouse, 205, route de Narbonne, 31077 Toulouse, France
2
Equipe Transformation du Pneumocoque, Laboratoire de Microbiologie et Gntique Molculaires, Universit de Toulouse, CNRS, 118, route de Narbonne , 31062 Toulouse, France Contact: Alexandre Stella (alexandre.stella@ipbs.fr)
Keywords: Proteins, peptides and small molecules quantification , Signaling, interactomics and post-translational modifications La transformation gntique est un processus de transfert horizontal de gnes caractristique du monde bactrien. Elle se droule en trois tapes distinctes : (i) la capture dADN double-brin prsent dans le milieu extrieur ; (ii) linternalisation dun brin dADN dans la cellule, couple la dgradation de son brin complmentaire ; (iii) lintgration par recombinaison homologue de lADN simple-brin (ADNsb) internalis. Une machinerie multiprotique spcialise, le transformasome est en charge du transport de lADN et de son intgration dans le gnome. La protine RecA, catalyseur de la recherche dhomologie et de lchange des brins dADN homologues, est une pice centrale de cet appareil spcialis de recombinaison dADN. Elle est assiste par plusieurs protines spcifiques du transformasome. Chez la bactrie Streptococcus pneumoniae, lun de ces effecteurs est la protine de liaison lADNsb : SsbB, dont lun des rles est de lier et protger lADNsb internalis. SsbB est un paralogue de la protine SsbA, universellement conserve chez les bactries et implique dans la plupart des mcanismes de maintenance de lintgrit du gnome. En plus de son activit de liaison lADNsb, SsbA agit dans ces processus comme plateforme dinteraction pour plusieurs protines des appareillages de la dynamique de lADN gnomique (1, 2). Afin de comprendre larchitecture du transformasome, et plus prcisment didentifier le rseau dinteraction spcifique entretenu autour de SsbB, nous avons engag ltude de son interactome en parallle de celui de SsbA. Pour cela nous avons adapt la mthodologie de capture de complexes protiques du Tap-Tag S. pneumoniae afin dextraire slectivement SsbB ou SsbA en association avec leurs partenaires en condition native. Dans une premire tape nous avons identifi par couplage nanoHPLC-MS/MS Orbitrap Velos lensemble des protines reprsentatives de ces complexes. Dans une deuxime tape, nous avons ensuite analys la dynamique de ces complexes en prsence dADN transformant. Cette analyse diffrentielle des protines partenaires a t ralise par des approches quantitatives label-free en utilisant le logiciel MFPaQ v4 (3). 1. Coste et al., PloS Genetics, 2010 2. Lecointe et al., EMBO J. 2007 3. Mouton-Barbosa et al., MCP, 2010
259/381
[P173] LC-MS/MS based urinary proteome analysis of obstructive nephropathy

Chrystelle Lacroix1, Ccile Caubet2, David Bouyssi1, Caroline Le Gall3, Benjamin Breuil2, Anne Gonzalez de Peredo1, Jean-Loup Bascands2, Bernard Monsarrat1, Joost Peter Schanstra2, Odile Burlet-Schiltz1
1
Laboratoire "Protomique et spectromtrie de masse des biomolcules", IPBS, CNRS UMR 5089, Universit de Toulouse, 205 Route de Narbonne, 31077 Toulouse, France
2
Laboratoire "Fibrose rnale - mcanismes et dtection", I2MC, INSERM UMR1048, Universit de Toulouse, CHU Rangueil -BP 84225 Institut Louis Bugnard, Btiment L4 Avenue Jean Poulhs , 31432 Toulouse, France
3
Equipe de Statistique et Probabilits, Institut de Mathmatiques, CNRS UMR 5219, Universit de Toulouse , 118 route de Narbonne , 31062 Toulouse, France Contact: Chrystelle Lacroix (chrystelle.lacroix@ipbs.fr)
Keywords: Clinical proteomics, Proteins, peptides and small molecules quantification , Bioinformatics et biostatistics dedicated to proteomics Dr Schanstras group has previously shown that urinary peptidome analysis by capillary electrophoresis coupled to mass spectrometry allows early prediction of obstructive nephropathy (ON) in newborns. Although these urinary peptidome biomarkers are of great potential clinical value, they are currently less informative on the pathophysiology of the disease. Therefore we decided to focus on the changes in the urinary proteome of ON patients using label-free LC-MS/MS and antibody-array analyses. Here, we present primarily the labelfree proteomic quantitative analysis. Urine samples from patients (n=5/group) were divided into 4 groups: bladder urine from controls (healthy), mild ON (NoOp), severe ON (Op) and pelvis urine from severe ON (Pyelon). 215 mL urine samples were concentrated and processed using the FASP based method (Wisniewski et al., Nat Methods, 2009). 30 g of protein was digested by trypsin. Peptide mixtures were then acidified and analysed 3 times (~4 g per injection) by online capillary LCMS/MS (Ultimate 3000 Dionex system coupled to a LTQ-Orbitrap-VELOS). Proteins were identified by MS/MS database search using Mascot and validation of identification results was performed with the MFPaQ v4 software (Bouyssi et al., MCP, 2007). Protein quantification was carried out with the label-free quantitative module of MFPaQ (Mouton-Barbosa et al., MCP, 2010). This module uses an identity-based method in which the precursor ion m/z and retention time of each identified peptide are retrieved from MS/MS database search result files, and used as a starting coordinate to extract the peptide elution peak intensity in the MS survey scans, allowing to perform peak detection in a robust and accurate way. Protein abundance was estimated by computing the sum of related peptides intensities. Such abundances were then normalized using the Probabilistic Quotient Normalization method (Dieterle et al, Anal Chem, 2006). Statistical analysis by Student t-test (corrected using Benjamini Hochberg adjustment for multiple testing) was performed to detect significant changes between two groups. Based on these comparisons we established a list of proteins that are specific for the obstructed or controlateral kidney. We are currently in the process of independent validation of a subset of these proteins. Two different approaches for validation are used: antibody-based validation (immunoblotting and ELISA) and multiple reaction monitoring on small (max 1.5 ml) urine samples to increase patient numbers during validation. The progress of this ongoing project will be shown.
260/381
[P174] Impacts de la variation gntique naturelle du protome de Saccharomyces cerevisiae et S. bayanus var. uvarum sur les principales voies mtaboliques
Mlisande Blein-Nicolas1, Warren Albertin1, Benot Valot1, Philippe Marullo2, Hao Xu4, Sylvie Huet4, Christophe Giraud5, Delphine Sicard1, Dominique de Vienne1, Michel Zivy1
1 2 3 4 5
UMR 0320 / UMR 8120 / PAPPSO Gntique Vgtale, Ferme du Moulon, 91190 Gif sur Yvette Universit Bordeaux 2, ISVV, USC nologie, 210 chemin de Leysotte, 33882 Villenave dOrnon SARCO/LAFFORT Group, 7 rue Franc Sanson, 33370 Floirac UMR MIA, Domaine de Vilvert, 78352 Jouy-en-Josas UMR CNRS 7641, Ecole Polytechnique, Route de Saclay, 91128 Palaiseau
Contact: Mlisande Blein-Nicolas (melisande.blein@moulon.inra.fr)
Keywords: Systems biology, Proteins, peptides and small molecules quantification Saccharomyces cerevisiae et S. bayanus var. uvarum sont deux espces de levure adaptes la fermentation alcoolique et dont l'utilisation en oenologie fait l'objet de nombreux dveloppements industriels. Afin d'analyser la variabilit gntique naturelle des abondances des protines de ces deux espces, nous avons slectionn 9 souches de S. cerevisiae et 6 souches de S. bayanus var uvarum isoles sur des substrats diffrents et dans des zones gographiques diverses, et les avons mises en culture anarobie dans du jus de raisin 18C. Aprs prlvement en phase stationnaire (3 rplicats indpendants par souche), le protome a t analys par LC-MS/MS et quantification sans marquage (logiciel MassChroQ1). Les bases de donnes de squence des deux espces ont t utilises pour l'identification des protines. Nous nous sommes tout d'abord intresss aux 3713 peptides prsentant des variations qualitatives. Aprs avoir limin les peptides de moindre abondance, nous avons isol 2399 peptides correspondant potentiellement des SAP (Single Amino-acid Substitution) qui nous ont permis d'obtenir une reprsentation de la variabilit gntique des souches. Nous avons ensuite estim les abondances de 622 protines partir de 5858 peptides rptables par une approche baysienne permettant d'inclure l'information porte par les peptides communs plusieurs protines. Au total, 382 protines prsentaient une variation gntique significative entre au moins deux souches, avec ratio d'abondance s'tendant entre 1.3 et 356. L'analyse des voies mtaboliques auxquelles ces protines appartiennent a permis de mettre en vidence i. des divergences entre espces concernant notamment les protines de la fermentation ainsi que les protines catalysant une mme raction. Ces divergences n'ont pas d'impact global sur la capacit fermentaire des deux espces; ii. des divergences entre souches de S. cerevisiae qui permettent de faire des hypothses sur les causes de leurs variations de capacit fermentaire. Certaines des souches de S. cerevisiae prsentant une mauvaise capacit fermentaire sont en effet caractrises par un niveau lev de protines impliques dans le mtabolisme nergtique et la rponse aux stress et des protines du mtabolisme des acides amins en moindre abondance. Globalement, l'ensemble de ces rsultats suggre que S. cerevisiae et S. bayanus var. uvarum se sont adaptes aux conditions de fermentation alcoolique par des mtabolismes diffrents.
261/381
[P175] Exploration du surfaceome de Listeria monocytogenes

Marie LAURIER1, Christophe CHAMBON2, Didier VIALA2, Mickal DESVAUX1, Michel HEBRAUD1
1
UR454 Microbiologie, Equipe QuaSA, INRA de Clermont-Ferrand, site de Theix, 63122 Saint-Gens Champanelle
2
Plate-Forme dExploration du Mtabolisme composante protomique, INRA de Clermont-Ferrand, site de Theix, 63122 Saint-Gens Champanelle Contact: Michel HEBRAUD (michel.hebraud@clermont.inra.fr)
Keywords: Systems biology Listeria monocytogenes est une bactrie pathogne Gram positif dorigine alimentaire. Elle est lagent tiologique de la listriose, une infection grave sous sa forme invasive (20 30% de mortalit), mais qui est relativement rare avec une incidence de 0,45 cas pour 100 000 habitants en France en 2008. Cette bactrie a la capacit de sadapter et de survivre dans divers environnements relativement hostiles, dadhrer et de former des biofilms sur des surfaces biotiques et abiotiques. Les protines de surface peuvent intervenir dans les mcanismes de perception et de rsistance lenvironnement, dans la virulence, la communication cellulaire ou encore dans ladhsion aux surfaces. Cest pour ces raisons quun intrt particulier est port aux protines de lenveloppe cellulaire, quelles soient membranaires ou paritales. La caractrisation de ce sous-protome, appel surfaceome, avec les approches classiques dextraction par fractionnement cellulaire et de sparation en gel dlectrophorse ne sont pas trs adaptes. En effet, ces approches ne permettent pas daccder la plupart des protines lies la paroi ou enchsses dans la membrane. Dautres stratgies de protomique, mises en uvre rcemment, tentent dapprhender les protines exposes la surface par des approches de marquage, danalyse soustractive ou de dcapage chimique ou enzymatique. Lobjectif de ce travail est de mettre au point une mthode danalyse des protines exposes la surface chez L. monocytogenes afin de lutiliser dans le cadre dtude comparatives souches mutantes/souche sauvage ou mode de croissance planctonique vs sessile, par exemple. Pour cela, une mthode de dcapage enzymatique a t teste en modulant diffrents paramtres (composition du tampon, dure dincubation, ajout dagent dnaturant) puis compare une mthode de dcapage chimique (Schaumburg et al., 2004). Les peptides extraits ont t prpurifis sur Sep Pak Light C18 (Waters) et analyss par LC-MS/MS, avec une colonne PepmapC18 (75 m x 150 mm, 2 m, 100 ), couple un spectromtre de masse nano-ESI-IT (LTQ Velos, ThermoFisher). Les protines ont t valides par la prsence dau minimum 2 peptides distincts. La prdiction de leur localisation a t effectue par plusieurs outils bioinformatiques, utiliss squentiellement pour caractriser la prsence dun peptide signal, le systme de scrtion utilis, la prsence dun motif de liaison lenveloppe et de domaines transmembranaires. Elle permet dvaluer lefficacit de chaque mthode extraire des protines de surface tout en limitant la contamination cytoplasmique. En effet, lun des principaux challenges de cette approche est de maintenir lintgrit des cellules bactriennes afin dviter une contamination massive par des protines cytoplasmiques. Les rsultats de cette tude mthodologique seront prsents et discuts.
262/381
[P176] Masschroq : a complete tool for mass spectrometry-based proteomic quantification

Edlira Nano1, Benot Valot1, Olivier Langella1, Mlisande Blein-Nicolas1, Ludovic Bonhomme1, Michel Zivy1
1 2
INRA, Plateforme d'Analyse Protomique de Paris Sud-Ouest, Ferme du Moulon, 91190 Gif-sur-Yvette, France CNRS, Plateforme d'Analyse Protomique de Paris Sud-Ouest, Ferme du Moulon, 91190 Gif-sur-Yvette, France
Contact: Edlira Nano (enano@moulon.inra.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics, Proteins, peptides and small molecules quantification MassChroQ (Mass Chromatogram Quantification) is a new software tool that performs XIC (eXtracted Ion Chromatogram) extraction, peak detection, alignment and quantification on data obtained from Liquid Chromatography-Mass Spectrometry (LC-MS) systems. A full description and evaluation of this tool can be found in [1]. MassChroQ is able to : - analyze data acquired by both high and low resolution mass spectrometers; - analyze data from label-free as well as from isotopic labeling experiments; take into account complex sample treatments as peptide or protein fractionation prior to LC-MS analysis. This versatility is mainly due to the fully configurable and traceable traits of the tool. MassChroQ is able to automatically and time-efficiently process large amounts of various data in one shot, its quantification results being ready to use for statistical analysis. In addition, it is open-source, every data it uses and produces is in open standard format (mzXML, mzML, CSV, gnumeric, ODT...) and its modular architecture allows easy integration in proteomic pipelines (like TPP or TOPP) or proteomic databases (like PROTICdb). We present here two studies of label-free and isotopic-labeling experiments where MassChroQ was used for quantification: - A study of genetic diversity in yeast (Blein-Nicolas et al, in prep). In this study, 9 strains of Saccharomyces cerevisiae and 6 strains of Saccharomyces bayanus var. uvarum (3 replicates of each) were analyzed without labeling by LC-MS/MS using an LTQ-Orbitrap (Thermo). From a total of 250Go of data MassChroQ quantified about 6684 peptides in 654 identified proteins in 30 minutes. 360 proteins were shown to vary significantly within and between species. - A quantitative phosphoproteomics study of in vivo changes during drought and recovery in the growing zone of maize leaves (Bonhomme et al, in prep). In this study, 40 maize leaf protein lysates were subject to dimethyl isotope labeling followed by SCX-fractionation. Ten fractions were recovered and used for further phosphopeptide enrichment using IMAC. The 400 resulting fractions were analyzed by LC-MS using an LTQ (Thermo) spectrometer. From a total of 450Go data, MassChroQ quantified 3659 phosphorylation sites accounting for 2462 distinct proteins. Statistical analysis of the quantification results evidenced fine localized phosphoproteomic changes possibly involved in the plant-growth process. For further information refer http://pappso.inra.fr/bioinfo/masschroq. to MassChroQ's homepage at
[1] Valot B, Langella O, Nano E, Zivy M . MassChroQ: A versatile tool for mass spectrometry quantification, Proteomics 2011, in press. Link: http://pappso.inra.fr/bioinfo/masschroq
263/381
[P177] A novel platform for comprehensive lipidomics screening based on MALDIQIT-TOF-MSn technology
Gerald STBIGER1, Wolfgang WERTHER2, Omar BELGACEM3
1 2 3
MEDICAL UNIVERSITY OF VIENNA, Spitalgasse 23, 1090 VIENNA (AUSTRIA) UNIVERSITY OF VIENNA, Doktor-Karl-Lueger-Ring 1, 1010 VIENNA (AUSTRIA) SHIMADZU KRATOS, Wharfside, Trafford Wharf Road, M17 1GP MANCHESTER (UK)
Contact: Omar BELGACEM (omar.belgacem@kratos.co.uk)
Keywords: Clinical proteomics Introduction Phospholipids (PLs) are essential components of biological membranes interacting dynamically to form platforms for protein activity, signal transduction and cell interactions. Changes in type, abundance and quantity of membrane PLs may be associated with the etiology of many diseases (e.g. neurological disorders, cancer, cardiovascular pathologies and atherosclerosis). Development of soft-ionization techniques (ESI- and MALDI-MS) facilitated the breakthrough for the analysis and understanding of the immense structural variability and functionality of membrane lipids. Efforts in the discovery of novel matrix substances, improvements of instrumentation, sensitivity and reproducibility open the possibility for setup of novel qualitative and quantitative MALDI-MS/MS lipidomics approaches. We introduce a comprehensive lipidomics screening platform based on MALDI-QIT-TOF-MSn combined with multivariate data analysis (MVDA) dedicated to clinical applications. Preliminary Data Several matrix substances have been evaluated for selective detection of different lipid classes. THAP and ATT were found the best matrices for detection of cationic PLs (e.g. LPC, PC, SM) and oxidized PLs (e.g. OxPCs), respectively. These lipid classes are exclusively detectable in positiveion mode either as [M+H/alkali]+ ions. Anionic lipid classes (e.g. PA, PE, PG, PI, PS) could be selectively detected in negative mode using 9-AA as the most universal or ATT as more selective matrix. The limit of detection of PLs was in the range <100 fmole on target allowing comprehensive profiling of the different PL-classes directly from mouse or human plasma (<110 nL equivalents) without need for any pre-separation. Using positive-ion mode verification of individual LPC, PC and SM species was possible via MS3 experiments from [M+Li]+ ions. OxPCs showed characteristic signature ions corresponding to degree of oxidative modification. In negative-ion mode [M-H]- ions provided conclusive structural information (e.g. carboxylate anions) in MS2 mode. This structural information was subsequently used for PL-profiling from biological samples based on statistical evaluation. Now ongoing work is centered towards implementation of the MALDI-QIT-TOF-MSn platform for clinical diagnostics. Novel Aspect MALDI-QIT-TOF-MSn is introduced as comprehensive lipidomics platform for basic research and clinical diagnostics in the future. Link: www.shimadzu.com
264/381
[P178] Analyse des polymres par spectromtrie de masse : apport du logiciel Polymerix
Valrie Bourdon1, Eric Leroy1, Olivier Thillaye du Boullay2, Fethi Bensaid2, Catherine Claparols1
1
Service Commun de Spectromtrie de Masse, Universit Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9
2
Laboratoire Heterochimie Fondamentale et Applique, Universit Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9
3
Laboratoire de Chimie de Coordination, CNRS, 205 route de Narbonne, 31077 Toulouse cedex 4
Contact: Valrie Bourdon (bourdon@chimie.ups-tlse.fr)
Keywords: High mass and high resolution mass Spectrometry Les polymres sont des macromolcules constitues dun enchanement dunits rptes appeles monomres. La grande varit de masse et de morphologie des polymres, ainsi que de nature des monomres dont ils sont constitus, permet dobtenir des matriaux trs divers avec de larges domaines dapplications. La caractrisation de ces matriaux est essentielle pour prdire et lucider les proprits et la morphologie des polymres. La spectromtrie de masse, plus particulirement la technique dionisation MALDI-TOF, est utilise pour ltude de composs non volatils de haute masse et est donc particulirement adapte lanalyse de polymres. La spectromtrie de masse permet dapporter un grand nombre dinformations au chimiste concernant la structure du polymre (extrmits de chanes, motif de rptition) et de dterminer sa masse molaire moyenne absolue. Cependant, linterprtation dun spectre de masse nest pas toujours vidente notamment lorsque lchantillon contient diffrentes familles. Cest l quintervient le logiciel Polymerix (Sierra Analytics) qui permet didentifier les polymres et de faire le calcul des grandeurs caractristiques : Ip, Mn, Mw, DPn, DPw pour chaque famille identifie. Il est utilisable avec les diffrentes marques de logiciels de masse et avec les diffrentes sources dionisation : ESI, MALDI-TOF Nous nous intresserons ici particulirement lanalyse MALDI. Un des principaux intrts de Polymerix consiste deisotoper les spectres de masse obtenus c'est--dire transformer le massif isotopique en un seul pic isotopique qui tient compte de labondance relative des lments prsents dans le polymre. Lutilit du logiciel sera aborde travers 3 exemples concrets : Un premier spectre MALDI dun chantillon polydisperse pour lequel le fait de deisotoper a pu mettre en vidence lenveloppe du polymre. Puis lexemple dun mlange de polymres avec un cart de masse de 2 uma. Les 2 polymres taient confondus dans un mme profil isotopique et la fonction deisotoping a permis de les diffrencier. Pour finir, un exemple o lanalyse du spectre MALDI combin avec le logiciel Polymerix a montr que la fonctionnalisation dun polyethylneglycol PEGXH par une molcule A ntait pas univoque. Nous avons pu dterminer que le polymre obtenu tait en fait un mlange de PEG de dpart (PEGXH), de PEG monofonctionalis (PEGX-A-H) et de PEG difonctionalis (PEGX-AAH). La RMN 1H ne permettait pas de lever lambigit quant la puret du polymre obtenu, monodisperse en analyse SEC.
265/381
[P179] Identification of heterodimers between the planar cell polarity receptors Vangl1 and Vangl2
Tania Guenneau-Puvijesinghe1, Edwige Belotti 1, Stphane Audebert2, Emilie Baudelet1, Luc Camoin1, Jean-Paul Borg1
1 2 3 4
Inserm U891, Centre de Recherche en Cancrologie de Marseille, 27, bd lei Roure, 13009 Marseille, France Institut Paoli-Calmettes, , 13009 Marseille, France Universit de la Mediterrane, , 13007 Marseille, France Marseille Proteomic Platform (MaP-IBIsa), ,
Contact: Stphane Audebert (stephane.audebert@inserm.fr) Keywords: Signaling, interactomics and post-translational modifications Neural tube defects (NTDs) are very common diseases in humans and rank second only to cardiac defects for inborn errors. The most common type of NTD is spina bifida whose prevalence in Europe is 5/10,000 births. Craniorachischisis is the most severe (and lethal) NTD characterized by a completely open neural tube along the whole body axis. At the molecular level, recent advances have highlighted that loss of function mutations in genes regulating planar cell polarity (PCP), a process required for the organization of epithelia sheets and morphogenesis of tissues, play a key role in the emergence of NTDs during early embryonic development. Genetic studies conducted in mouse models that were recently confirmed in humans have revealed that mutations in the cell polarity receptor Vangl2 cause PCP defects and craniorachischisis. Vangl2 is a 521-amino acid membrane protein with poorly described functions and is composed of four putative transmembrane domains. Interestingly, Vangl2 genetically interacts with Vangl1, a close homolog, as double heterozygous Vangl1/Vangl2 mice develop identical PCP defects and craniorachischisis as Vangl2-/- deficient mice. It remains unknown whether these proteins belong to a common protein complex or act in parallel pathways. Considering the very close similarity between the peptide sequences of Vangl1 and Vangl2, the study of these proteins is severely hampered by the lack of antibodies able to biochemically discriminate between the two homologs. In this study, we have generated a highly specific monoclonal anti-Vangl2 antibody able to efficiently purify Vangl2 from protein extracts. The specificity of the antibody was demonstrated in western blot and immunoprecipitation assays. We screened breast cancer cell lines and identified SKBR7 as a cell line expressing both Vangl1 and Vangl2. We then used an immunoaffinity-based experimental approach using the anti-Vangl2 antibody to isolate endogenous Vangl2 and its associated binding partners from SKBR7 protein extracts. Protein analysis of the immunoprecipitated proteins was undertaken using denaturing SDS-PAGE separation, in-gel trypsin digestion and Orbitrap mass spectrometry analysis. We efficiently identified endogenous Vangl2 with very good peptide sequence coverage and through the analysis of post-translational modifications confirmed the methionine encoded by the predicted start codon as acetylated. Analysis of proteins present in Vangl2 immunoprecipitates showed that Vangl2 copurifies with endogenous Vangl1. Using GFP or Myc-tagged Vangl1 and Vangl2 constructs, we further confirmed that these cell polarity receptors are able to form homo- and hetero-complexes in living cells. The data presented here demonstrate that Vangl1 and Vangl2 form protein complexes and therefore establish a paradigm for the understanding of the morphogenetic defects in which these proteins are implicated. Link: http://crcm.marseille.inserm.fr/
266/381
[P180] Pre-treatment of proteins before enzymatic digestion: a critical step to consider further in proteomic analyses.
Sylvie Kieffer-Jaquinod1, Mathieu Baudet1, Alexandra Kraut1, Maighread Gallagher-Gambarelli1, Michel Jaquinod1, Christophe Bruley1, Yohann Cout1
1
Biologie Grande Echelle, CEA / INSERM 1038 / UJF, 17, rue des Martyrs, 38054 Grenoble cedex 9, France
Contact: Yohann Cout (yohann.coute@cea.fr)
Keywords: Separative analysis methods In bottom-up proteomic studies, after optional biological or biochemical fractionations, extracted proteins are classically submitted to specific treatments before enzymatic digestion and analysis of the generated peptides using mass spectrometry. These pre-digestion steps mainly aim at disassembling disulfide bonds and blocking free sulfhydryl groups of cysteines, rendering proteins more accessible to enzymatic cleavages and facilitating unambiguous identification of cysteine-containing peptides. However, even sounding straightforward, these procedures need to be fully controlled in order to avoid irreproducible amino acid modifications as well as complexification of the studied proteomes. In the presented work, we evaluated two different ways of preparing proteins from various complex samples of different origins before in-gel digestion: reduction/alkylation and hydrogen peroxide oxidation. Number of identified peptides as well as amino acids (notably C, M) representations and modifications were studied. Our results highlighted the strengths and weaknesses of both protocols; for example more cysteine-containing peptides were identified using the reduction/alkylation strategy compared to the hydrogen peroxide oxidation whereas an opposite observation was made for methionine-containing peptides. We then worked on the optimization of both protocols. For the oxidation protocol, performic acid was tested using several concentrations of acid and of hydrogen peroxide. This led to an increase in the number of identified peptides, the conversion of all methionines to a unique and maximal oxidation state but also to the disappearance of tryptophan-containing peptides from the list of identified peptides. For the reduction/alkylation protocol, several concentrations of alkylating reagents as well as the addition of a step stopping alkylation were tested. Conditions generating maximal peptide identifications and minimal generation of side reactions were found. The improvements made in the reduction/alkylation protocol were also successfully tested with in-solution trypsin digestion. In conclusion, pre-treatment of proteins before digestion is a critical step that has to be carefully studied. Reduction/alkylation and mild oxidation of proteins are complementary protocols that can be used in parallel to further characterize proteomes of interest.
267/381
[P181] Caractrisation par spectromtrie de masse Maldi-Tof de polyisoprnes structure complexe

Christelle Absalon1, Samira Ouardad2, Christiane Vitry1, Claire Mouche1, Patricia Castel1, Alain Deffieux2, Frdric Peruch2
1 2
ISM-CESAMO Universit Bordeaux1 UMR 5255, 351 cours de la Libration, 33405 Talence LCPO Universit Bordeaux 1 UMR 5629, 16 avenue Pey Berland, 33607 Pessac
Contact: Christelle Absalon (c.absalon@ism.u-bordeaux1.fr)
Keywords: Systems biology Le caoutchouc naturel, un poly(isoprne) de structure purement 1,4 cis prsentent des proprits ingales par ses homologues synthtiques. Pour cette raison la production mondiale de caoutchouc naturel demeure stratgique et est toujours proche de 10 millions de tonnes, soit 50% de la demande mondiale annuelle en lastomres. Alors que les voies de synthse dveloppes jusqu prsent pour accder des homologues du caoutchouc naturel sont bases sur la polymrisation par voie anionique ou par coordination de lisoprne, lapproche suivie par la nature pour synthtiser les polyterpnes est base sur la polymrisation caractre cationique dun autre monomre, lisopentnyl pyrophosphate (IPP) qui est la brique de construction universelle de la famille des terpnes. Nous avons alors dvelopp une voie cationique pour polymriser des modles de lIPP afin daccder des polyterpnes approchant la structure des produits naturels. Les polyisoprnes obtenus par voie cationique possdent une structure relativement complexe du aux nombreuses ractions secondaires engendres par ce type de polymrisation (amorage parasite, branchement, cyclisation, ). Les analyses par rsonnance magntique nuclaire et par analyse par chromatographie dexclusion strique SEC ne nous ont pas permis de mettre en vidence lensemble de ces ractions secondaires. En revanche, lanalyse de spectromtrie de masse Maldi-TOF sest rvle tre un outil performant permettant de mettre en vidence diffrents types damorage par exemple. Lobjet de cette tude est de proposer une mthode optimise pour la caractrisation de ces polyisoprnes. En premier lieu, le choix de la matrice et des sels dionisation savrent particulirement primordial pour lobtention de spectres homognes et reproductibles. Cette technique danalyse a pu non seulement fournir des informations sur les mcanismes mis en jeu mais aussi une quantification relative des familles.
268/381
[P182] Conversion of fern (Pteris vittata L.) biomass from a phytoremediation trial in sub- and supercritical water conditions : qualitative and quantitative study by GC-MS
Christelle Absalon1, Marion Carrier2, Marie-Hlne Lescure1, Anne Loppinet-Serani2, Cyril Aymonier2, Michel Mench3
1 2 3
ISM-CESAMO Universit Bordeaux 1 UMR 5255, 351, cours de la Libration, 33405 Talence ICMCB CNRS Universit Bordeaux 1 UPR 9048, 87, avenue du Dr. A. Schweitzer, 33608 Pessac UMR BIOGECO INRA 1202 Universit Bordeaux 1, avenue des Facults, 33405 Talence
Contact: Christelle Absalon (c.absalon@ism.u-bordeaux1.fr)
Keywords: Systems biology Unlike organic compounds, trace elements in contaminated soils cannot be degraded. To reduce pollutant linkages evidenced by risk assessment, the clean-up of many contaminated sites requires removing trace elements in excess from the soil. Phytoextraction refers to the uptake and translocation of soil contaminants by plant roots into generally the above ground plant parts to remove contaminants from soil and promote long term soil cleaning. This natural, solar-energy driven phytoremediation option does not compromise the topsoil functioning and fertility and may improve it, without secondary pollution, huge costs, and consummation of primary raw. Chinese brake fern (Pteris vittata L.), an arsenic (As) hyperaccumulator is an option to phytoextract As in contaminated soils. Post-harvest disposal of such trace elementcontaminated plant biomass is a key-point to gain financial returns from phytoextraction and to support public, policy maker and stakeholder acceptance. An innovative process using sub and supercritical water treatments propose the complete oxidation of biomass or its conversion into valorizable products via gasification or liquefaction mechanisms. This work deals with characterization of obtained products in liquid phase. The chemistry of biomass degradation has been studied with plant model compounds such as cellulose, sugars, and lignin and subsequently numerous compounds such as phenols, furfurals, carbon acids, sugars and condensation products have been detected after a supercritical treatment. Therefore key organic compounds, i.e. phenol, cyclopentenones, furfural, and guaacols, were measured by GC-MS in the liquid phase following the sub- and supercritical treatments. In this study, we will follow the influence of the temperature during supercritical water treatments on the formation of organic compounds. Qualitative and quantitative study of liquid phases will help understanding degradation mechanisms.
269/381
[P183] Dveloppement dune approche multi-plateforme pour lanalyse des phytomicronutriments et de leurs mtabolites chez lhomme
Noura Meklat1, Mercedes Quintana2, Bernard Lyan1, Claudine Manach2, Estelle Pujos-Guillot1
1
Plateforme d'Exploration du Mtabolisme, Centre de Recherche de Clermont-Ferrand/Theix, 63122 SaintGenes-Champanelle

2
UMR1019 Nutrition Humaine, Centre de Recherche de Clermont-Ferrand/Theix, 63122 Saint-GenesChampanelle Contact: Noura Meklat (claudine.manach@clermont.inra.fr)
Keywords: Instrumentation, Proteins, peptides and small molecules quantification Le Food metabolome est lensemble des mtabolites issus de lalimentation et retrouvs dans les fluides biologiques en particulier le sang et lurine (Walsh et al.,2006 ; Manach et al., 2009, Van Duynhoven et al., 2011). Les phytomicronutriments constituent un ensemble htrogne de composs appartenant diverses familles chimiques tels que les polyphnols, les terpnes, les phytostrols, les carotnodes etc. Mme sils ne font pas partie des nutriments essentiels, leurs effets sur la sant sont de plus en plus tudis. De plus ils peuvent constituer des biomarqueurs de consommation des fruits et lgumes, qui permettront de mettre en vidence les relations entre les habitudes alimentaires et diverses pathologies dans les tudes pidmiologiques. Lobjectif de ce travail est le dveloppement dune stratgie analytique permettant de couvrir un maximum de la composante phytomicronutriments du Food metabolome , et pouvant tre applique lanalyse dchantillons urinaires et plasmatiques de consommateurs de fruits et lgumes issus dune cohorte. Un inventaire des familles de phytomicronutriments prsents dans les aliments a t ralis. Lexamen dtaill des proprits physico-chimiques de chacun de ces composs et de leurs mtabolites, nous a permis de slectionner 28 composs reprsentatifs des diverses familles. En raison du large ventail de masses, de polarits et de fonctions chimiques mises en jeu et pour rendre lanalyse de ces mtabolites la plus exhaustive possible nous avons mis en place une stratgie analytique combinant la fois la chromatographie gazeuse couple la spectromtrie de masse (GC-EI/MS) et la chromatographie liquide couple la spectromtrie de masse (LCESI/APCI-MS mode positif et ngatif). Les conditions chromatographiques, les mthodes de dtection, ainsi que les tapes de prparation dchantillons ont t optimiss dans le but garantir la couverture analytique de la mthode, tout en limitant la variabilit induite. Nous prsentons ici les premiers rsultats obtenus sur matrices urinaires et plasmatiques supplmentes avec les standards des composs slectionns comme reprsentatifs des principaux phytomicronutriments du Food metabolome .
270/381
[P184] Localization of the active site of Abf2, a mitochondrial DNA repair protein
Guillaume GABANT1, Franoise CULARD1, Stphane GOFFINONT1, Bertrand CASTAING 1, Martine CADENE1
1
Centre de Biophysique Molculaire, CNRS UPR4301, rue Charles Sadron, 45071 Orlans, France
Contact: Martine CADENE (martine.cadene@cnrs-orleans.fr)
Keywords: Signaling, interactomics and post-translational modifications Mitochondrial genomes are constantly damaged by endogenous reactive oxygen species generated during the electron transport chain of cellular respiration. These DNA lesions are potentially mutagenic and cytotoxic and most of them are corrected through the base excision repair (BER) pathway. Autonomously replicating sequence (ARS)-binding factor 2 (Abf2) protein is essential for the maintenance of mitochondrial DNA of Saccharomyces cerevisiae. We have recently shown that Abf2 exhibits an abasic (AP) site cleavage activity. Here we investigate the amino acid responsible for this protein activity. In these experiments we used the intermediate of the enzymatic reaction which involves a Schiff base formation between the enzyme and DNA that can be stabilized by a sodium borohydride reduction to form a covalently linked complex. Mass spectrometry characterization of protein-DNA or peptide-nucleic acid heteroconjugates is hampered by the contrasting physical-chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Furthermore, the oligonucleotide part of the heteroconjugate had to be small enough to ensure that the species under investigation exhibit peptide-like properties to facilitate peptide analysis and sequencing in the positive ion mode. A two-step enrichment method (avidin-biotin affinity chromatography and metal chelate chromatography) combined with enzymatic degradation of protein (Trypsin proteolysis) and oligonucleotide (Nuclease P1 hydrolysis) prior to mass spectrometric analysis was developped. Matrix-assisted laser desorption/ionization mass spectrometry experiments on proteolytic peptides allow for the identification of a modified sequence containing three potential targeted residues: Lys147, Lys156 and Lys158. Sequencing of the corresponding peptidenucleic acid heteroconjugate by nanoelectrospray ion trap MS/MS mass spectrometry shows that Lys147 is not modified but cannot discriminate between Lys156 and Lys158 which are in close proximity. Additional proteolysis of the tryptic products with endoproteinase Glu-C (V8 Protease) leads to cleavage at Asp157 with unambiguous assignment of Lys158 as a modified residue. Directed mutagenesis experiments are underway to confirm this result.
271/381
[P185] Proteinscape, un outil convivial et performant au service de la protomique : de la gestion des projets lexploitation des donnes de spectromtrie de masse
Lauriane Kuhn1, Pierre-OIivier Schmit2, Yannis Franois3, Emmanuelle Leize-Wagner3, Philippe Hammann1
1
Plate-forme Protomique Strasbourg Esplanade, Institut de Biologie Molculaire et Cellulaire, FRC1589 , 15 rue Ren Descartes , 67084 Strasbourg Cedex
2 3
Bruker Daltonique, 34 rue de lIndustrie, BP10004, 67166 Wissembourg Cedex
Laboratoire de Dynamique et Structure Molculaire par Spectromtrie de Masse, UMR7177, Facult de Chimie, 1 rue Blaise Pascal, 67008 Strasbourg Cedex Contact: Lauriane Kuhn (proteomic-esplanade@unistra.fr) Keywords: Systems biology Grce la mise en place de mthodes danalyse protomique haut-dbit , les plates-formes technologiques en protomique peuvent traiter un nombre important et croissant de demandes danalyse. Mais il y a une contrepartie: savoir stocker le volume de donnes gnres et savoir exploiter ces donnes de manire donner un sens aux identifications et rpertoires protiques obtenus. Ce constat a permis de prendre conscience de limportance des outils informatiques et bioinformatiques, indispensables chaque tape du traitement des chantillons. Notre plate-forme danalyses protomiques, situe Strasbourg Esplanade, ralise des prestations de service pour lanalyse des protines et peptides par spectromtrie de masse. De part laugmentation du nombre de demandes danalyse, de la multiplicit des approches utilises (1D- et 2D-E, (LC)-MALDI-TOF/TOF et nanoLC-MS/MS) et de la profondeur danalyse souhaite (identification, modification, quantification), nous avons opt pour la solution logicielle Proteinscape (Bruker Daltonique). Cet outil, convivial et performant, nous assiste quotidiennement sur la plate-forme, et sarticule autour des tapes cls suivantes: 1)Gestion des chantillons et des projets : Proteinscape nous permet de hirarchiser de manire intelligente les prestations de service et les projets, selon la granulomtrie souhaite (projet / chantillon / gel / sparation / enzyme / donnes MS multi-constructeurs / etc). La manire dutiliser cette plate-forme logicielle nous garantit une traabilit des donnes et leur stockage dans un outil de type LIMS (protocoles, scans de gels, recherche dans les banques de donnes, fichier contenant les protines valides, partage des donnes entre plusieurs utilisateurs agres ). 2)Recherches dans les banques de donnes : Proteinscape permet une utilisation complmentaire des moteurs de recherches Mascot et Phenyx, tape lissue de laquelle une validation, automatique ou manuelle, des identifications et une valuation de la qualit de donnes (FPR) est ralise. 3)Exploration des donnes : Proteinscape reposant sur une base de donnes SQL sous-jacente, nous utilisons des requtes afin dextraire les informations les plus pertinentes des analyses effectues (interrogation de plusieurs chantillons pour rechercher une protine ou un peptide dintrt, mise en vidence de protines diffrentiellement exprimes par comparaison de diffrentes conditions dtudes, ). 4)Exports : dans le cadre du rendu de rsultats par la plate-forme, lutilisation de Proteinscape savre indispensable pour ldition de rapports danalyses contenant les dtails des identifications obtenues sur un ou plusieurs chantillons. De plus, Proteinscape permet galement deffectuer des exports de donnes utilisables pour la valorisation des rsultats et le respect des guidelines des journaux de protomique. Link: http://www.bdal.com/products/software/proteinscape/overview.html
272/381
[P186] CESI-MS. Principe et application pour l'analyse sensible de mlanges peptidiques complexes
Michael Biacchi1, Nina Pourhassan1, A. A. Heemskerk3, B. Schoenmaker3, Yannis Francois1, Emmanuelle LeizeWagner1, Jean-Marc Busnel3, O.A. Mayboroda3
1
Laboratoire de Dynamique et Structure Molculaire par Spectromtrie de Masse (LDSM2), CNRS UMR 7177, Institut de Chimie de Strasbourg, 1 rue Blaise Pascal, 67000 Strasbourg, France
2 3
Laboratoire Biochimie Toxicologie, CHR Metz Thionville, 2 rue de Friscaty, 57126 Thionville, France
Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, Pays-Bas
4
Beckman Coulter Inc., 250 South Kraemer Boulevard, CA 92821-6232 Brea, Etats-Unis
Contact: Yannis Francois (yfrancois@unistra.fr)
Keywords: Instrumentation, Clinical proteomics La nouvelle plateforme CESI, reposant sur la technologie OptiMS de la socit Beckman Coulter a t rcemment mise en place au laboratoire. Cette technologie, base sur lutilisation dun capillaire de sparation dont une extrmit a t traite par acide fluoridrique a pour but de permettre un couplage idal de llectrophorse capillaire (CE) et de la spectromtrie de masse par lectronbulisation (ESI-MS). Dans un premier temps, afin de caractriser cette nouvelle interface, diffrentes expriences ont t ralises afin de dterminer quelle tait la gamme de dbits compatible avec la technologie OptiMS. Il a ainsi t dmontr que cette approche permettait de gnrer un spray stable pour des dbits allant de plusieurs centaines de nL/min a des valeurs inferieures a 10 nL/min, ceci permettant de tirer profit du rgime nanoESI (< 30 nL/min) qui se caractrise par une augmentation de la sensibilit et une diminution du phnomne de suppression dions. Par la suite, lanalyse de mlanges peptidiques de complexit croissante a dmontr que cette nouvelle plateforme fournit dexcellentes sensibilits permettant la dtection de faibles quantits dchantillon avec un pouvoir rsolutif optimal. De plus, il est ici dmontr quune tape de prconcentration par isotachophorse transitoire (tITP), permettant daugmenter significativement le volume dchantillon inject tout en conservant lefficacit et la rsolution des pics, est parfaitement compatible avec cette nouvelle interface. Les limites de concentrations et de dtections obtenues sur ce systme sont de lordre du subnanomolaire et du nanomolaire selon lamplitude de la phase de preconcentration.
273/381
[P187] Dveloppement dune approche protomique chez le crabe Carcinus maenas (L.) : mise au point dun protocole dextraction de protines partir des branchies et rvlation des protines ubiquitinises
Franois Panchout1, Julie Letendre1, Xavier Denier1, Florence Bultelle1, Batrice Rocher1, Franois Leboulenger1, Fabrice Durand1
1
Laboratoire dEcotoxicologie-Milieux Aquatiques (LEMA) EA3222, IFRM 23, Universit du Havre, 25 rue Philippe Lebon B.P. 540, 76058 Le Havre, France Contact: Batrice Rocher (beatrice.rocher@univ-lehavre.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications Lacclimatation des organismes soumis des contaminations chimiques est devenue une question majeure au vu de la pollution croissante et complexe qui touche les cosystmes. Les espces vivant en milieux ctiers sont exposes de faon chronique un stress physique et chimique. Parmi ces espces, le crabe, Carcinus maenas, prsente une large rpartition gographique, occupe des habitats varis de lembouchure des estuaires jusquau circalittoral des mers ctires, traversant des continuums hydrologiques salinit, temprature, oxygnation et dure dexondation variables, dmontrant ainsi une forte plasticit physiologique et suggrant des capacits dacclimatation et de rsistance importantes. En tant que consommateur privilgi de bivalves estuariens et mdiolittoraux (moules, tellines et scrobiculaires), Carcinus maenas est aussi un modle potentiel de bioamplification des contaminants. Lobjectif de ltude est dvaluer la rsistance de crabes Carcinus maenas des conditions de stress contrles en fonction de leur trait de vie, en particulier en fonction de lexposition la pollution pralablement subie. Pour se faire, une approche ouverte a t dveloppe, qui vise mettre en vidence des acteurs protiques dont lexpression traductionnelle, voire posttraductionnelle, est modifie sous limpact des facteurs environnementaux et/ou sous leffet de la contamination chimique. Cette tude a pour but, terme, dlucider des mcanismes molculaires et cellulaires impliqus dans la rsistance aux polluants. Lanalyse sera ralise par comparaison des profils 2DE obtenus aprs coloration au nitrate dargent et aprs transfert sur membrane et immunodtection des protines polyubiquitinises. Lubiquitination est une modification post-traductionnelle qui oriente les protines vers le protasome en vue de leur protolyse. Ce systme complexe demeure peu tudi chez les invertbrs aquatiques. Le tissu choisi, la branchie, occupe une position privilgie linterface entre le reste de lorganisme et le milieu extrieur. Cependant, les branchies des dcapodes sont recouvertes dune cuticule de chitine qui rend inefficace les protocoles classiques dextraction et de solubilisation des protines. De ce fait, le travail prsent ici est centr sur le dveloppement de la technique dextraction et de solubilisation des protines dans les branchies chez le crabe Carcinus maenas. Les rsultats en termes de qualits des empreintes protomiques obtenues aprs 2DE indiquent que la mthode la plus efficace et reproductible consiste en une extraction par broyage dans lazote liquide suivie dune prcipitation au trichloroactate avant solubilisation. Le marquage des protines ubiquitinises montre une grande htrognit des profils au sein de la population de crabes teste avec une protine majoritairement marque et qui reste encore identifier.
274/381
[P188] Fast Analysis of Mineral Oils in Recycled Cereal Food Packaging using Direct Sampling Analysis Time-of-Flight Mass Spectrometry, DSA-TOF MS
Sean Daugherty 1, Massimo Santoro1, Shida Shen1, Karima BAUDIN2
1 2
PerkinElmer Inc, 710 Bridgeport Avenue, CT 06484 Shelton, USA Perkin Elmer France, 16 Avenue du Quebec, 91140 Villebon sur Yvette, France
Contact: Karima BAUDIN (karima.baudin@perkinelmer.com)
Keywords: Instrumentation, Organic and inorganic mass spectrometry NOVEL ASPECT A fast and sensitive method for determination of mineral oils in recycled food packaging using Direct Sample Analysis Time-of-Flight Mass Spectrometry (DSA-TOF MS). INTRODUCTION Recent tests on recycled food packaging have found a variety of mineral oils, some associated with components of printing ink. Mineral oil consists of various hydrocarbons known as mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). The safe upper limit for MOSH according to the UN Food and Agriculture Organization/World Health Organization Joint Expert Committee on Food Additives is 0.6 mg/kg. This level is often exceeded because of presence of mineral oil from newspaper and other printed paper utilized in the recycling process. METHODS Measurement was performed using a PerkinElmer TOF MS system equipped with a PerkinElmer Direct Sample Analysis (DSA) ion source. The DSA source ionizes compounds evaporated from the surface and bulk cardboard packaging through gas phase charge exchange reactions. It allows detection, identification, spatial mapping of migration, and relative abundance determination of mineral oils in the packaging material without sample preparation. PRELIMINARY DATA A fast DSA method for the determination of mineral oils in recycled packaging allows monitoring the progress of the printing inks through the recycled matrix, determination of the different mineral oils present, and their concentrations. Data acquired from a coffee shop cardboard cup holder in positive ion polarity mode using the DSA source detected peaks separated by m/z 28, indicating a hydrocarbon chain with an increasing number of C2H4 units.
275/381
[P189] Tissue proteomics for ovarian epithelial cancers biomarkers hunting: screening and targeting for new issues in biology and pharmacology
Rmi Longuespe1, Charlotte Boyon1, Olivier Kerdraon4, Annie Desmons1, Robert Day2, Denis Vinatier3, Robert Day2, Isabelle Fournier1, Michel Salzet1
1
Laboratoire de Spectromtrie de masse fondamentale et applique, Universit des Sciences et Technologies de Lille, 59650 Villeneuve D'Ascq, France
2 3 4
Institut de Pharmacologie de Sherbrooke, Universit de Sherbrooke, J1H5N4 Sherbrooke, Canada (Qubec) Service de chirurgie gyncologique, CHRU Lille, 59000 Lille, France Centre d'Anatomie et de Cytologie pathologiques, CHRU Lille, 59000 Lille, France
Contact: Rmi Longuespe (r.longuespee@gmail.com)
Keywords: Imaging mass spectrometry, Clinical proteomics Direct analysis of tissues by MALDI-MS is an interesting strategy for markers hunting in pathologies by giving molecular information at the tumor level. Using different chemical preparations, benign and malignant ovarian biopsies were analyzed by MALDI-TOF-MS for peptides, proteins, and high-mass proteins, in automatic profiling assays to investigate specific signatures for diagnosis. By this strategy, coupled to bottom-up procedures, several biomarkers from ovarian carcinoma regions were obtained and classified as proteins associated with cell proliferation, immune response modulation, signaling to the cytoskeleton, tumor progression, and epithelial-tomesenchymal cell transformation. These specific biomarkers were then validated by immunocytochemistry using Tag-mass technology, Western blot, and PCR. One of these markers is a fragment of the immunoproteasome-11S which was found in borderline and stage-I serous, endometrioid, and mucinous tissues. This makes it a potential target for wide screenings of the early development of the disease and reveals an early biological process of immunotolerance for cancer cells in the ovarian environment. Then, our recent developments allowed us to associate immunocytochemistry and MSI to track this molecule in biopsies by imaging and quantifying the tryptic peptides of anti-Reg Alpha antibodies. This strategy could be used for monitoring of the effect of treatments of the pathology by screening the evolution of the presence of the compound in malignant tissues. All these assays clearly demonstrate the use of the technique for the understanding of cancers mechanistic and pave a new way for new pharmacologic applications.
276/381
[P190] Quantitative analysis of proteasome inhibitor effect in acute myeloid leukaemia cells
Mariette Matondo1, Marlne Marcellin1, Karima Chaoui1, Marie-Pierre Bousquet-Dubouch1, Sandrine Uttenweiler-Joseph1, Ruedi Aebersold2, Bernard Monsarrat1, Odile Burlet-Schiltz1
1 2
Institut de Pharmacologie et de Biologie Structurale , 205 route de Narbonne, 31077 Toulouse Institute of Molecular Systems Biology, Wolfgang-Pauli-Str. 16, 8093 Zurich
Contact: Mariette Matondo (matondo@imsb.biol.ethz.ch)
Keywords: Systems biology, Proteins, peptides and small molecules quantification The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in cell cycle control and signal transduction. Aberrant regulation of cell cycle proteins can result in accelerated and uncontrolled cell division, leading to tumorigenesis and cancer1. Proteasome inhibitors possess potent antitumor activity and have been used against a broad spectrum of human tumor types2-4. However, the effect of these compounds at the molecular level remains unclear. In the present study, we have investigated the effect of proteasome inhibitors on human acute myeloid leukaemia (AML) cells. We demonstrated that exposure to several proteasome inhibitors (MG132, PS-341 and Lactacystin) induced a higher level of apoptosis in immature KG1a cells in comparison to mature U937 cells5. To go further into the understanding of the effect of proteasome inhibitors on these cell lines, we investigated their protein expression profile before and after proteasome inhibition using SILAC quantitative proteomic analyses. Cell fractionation into cytoplasmic and nuclear extracts was used to reduce sample complexity and maximize protein coverage. The proteins were then separated on a 1D SDSPAGE gel and digested. Mass spectrometric analysis was performed on both LTQ-Orbitrap XL and Velos instruments. To evaluate the reproducibility and relevance of the variations, two biological replicates were performed. Overall, more than 7,000 proteins were accurately identified and quantified across the samples, where 350 proteins were significantly regulated upon proteasome inhibition in both KG1a and U937 cells. Some of these regulated proteins will be quantified by SRM in cells treated under multiple relevant conditions to validate further their role in the differential apoptotic responses to the proteasome inhibition. References 1-C.Wojcik and al., J cell.Mol.Med 2002 2- J. Adams and al., Cancer Research 1999 3- Lu M and al., J Cell Biochem. 2006 4- Szokalska A and al., Cancer Research 2009 5-Matondo and al., Leukemia Research 2010
277/381
[P191] Club-jeunes de la Socit Franaise d'Electrophorse et d'Analyse Protomique (cjSFEAP)

Rmy Aurand1, Barbara Deracinois1, Erwann Arc1, Sarah Lennon1, Paul Morel1, Mathieu Trauchessec 1, Stphanie Petiot 1, Morgane Couvet1, Bertrand Delaunois1, Cline Boursier1
1
cjSFEAP, Club-Jeunes de la Socit Franaise d'Electrophorse et d'Analyse Protomique, 10 rue Vauquelin, 75005 Paris Contact: Cline Boursier (celine.boursier@u-psud.fr)
Keywords: Separative analysis methods, Proteins, peptides and small molecules quantification , Bioinformatics et biostatistics dedicated to proteomics Le Club-Jeunes de la SFEAP, ou cjSFEAP, est, comme son nom l'indique, la filire jeune de la SFEAP. Son objectif est de former un rseau de relations entre les jeunes protomistes pour qu'ils puissent changer leurs expriences et s'entraider pour trouver des solutions aux diffrents problmes rencontrs mais aussi pour prparer leur insertion professionnelle et les former dans le domaine de la protomique. Pour cela, des rencontres scientifiques sont organises tous les ans par le cjSFEAP : - "Les journes du Club-Jeunes" qui ont lieu en mai-juin et qui regroupent une trentaine de jeunes et une dizaine d'intervenants sniors ; - "La session Jeunes" lors du congrs annuel, o les membres prsentent leurs travaux. Pour tre membre, il faut remplir les conditions suivantes : - tre adhrent la SFEAP ; - tre tudiant et/ou avoir moins de 35 ans ! Le Club-Jeunes compte ce jour prs de 80 membres. Pour se renseigner sur l'actualit du cjSFEAP, il suffit de se connecter au site web : http://cj.sfeap.fr Vous pouvez galement nous contacter via notre mail : club-jeunes@sfeap.fr Nous proposons l'ensemble des jeunes travaillant dans le domaine de la protomique de nous rejoindre pour largir notre rseau. Link: http://cj.sfeap.fr
278/381
[P192] Multimodal size and charge based analysis of the egfr signaling pathway using an automated capillary-based system for nanoscale protein analysis
Francisco Ramirez1 , Irina Kazakova1 , Jessica Dermody 1 , Thierry Salomon1, Tom Yang1 , Uyen Nguyen1 , Robert Gavin1 , Annegret Boge1
1
ProteinSimple (Cell Biosciences Inc.), 3040 Oakmead Village Drive, 95051 CA Santa Clara USA
Contact: Thierry Salomon (thierry.salomon@proteinsimple.com)
Keywords: Signaling, interactomics and post-translational modifications, Clinical proteomics, Instrumentation Background: Aberrant expression and signaling of epidermal growth factor receptor (EGFR) is a common occurrence in a variety of cancers including breast cancer. Understanding how EGFR signaling impacts disease progression is key to the development of novel therapeutics. Analysis of EGFR expression in cancer samples frequently employs Western blot analysis. However, in order to fully understand signaling events, evaluating changes in signaling proteins is accomplished utilizing size-based as well as charge-based separation techniques, each of which is followed by immunoassay detection. Methods: To this end, we have developed the NanoPro2 (NP2), which combines the ability to perform size- and isoelectric charge-based separation of proteins in nanoscale volumes in one instrument. The NP2 performs these Capillary Electrophoresis (CE) modes coupled to an automated workflow that eliminates the need for manual processing of multiple steps. Proteins from a single sample preparation were analyzed in two assay modes: either in a native conformation using charge-based separation or in a denatured state followed by size-based separation. Results: Utilizing both modes allowed for detailed analysis of post-translational modifications (isoelectric charge-based separation) and protein abundance (size-based separation) within a prepared sample. As a validation of the dual-mode capabilities of the NP2, we investigated EGFR signaling in response to treatment with EGF. Conclusion: We showed that the same cellular lysate preparation could be used for analysis by charge and size-based separation, thus establishing a highly flexible system that utilizes a single lysate for multiple assay modes. Advantages of the NP2 assay over slab gel-based assays shown include ease of use, minimal user intervention, automatic analysis and excellent reproducibility. Link: http://www.proteinsimple.com/
279/381
[P193] Caractrisation de peptides lasso par EPD et EDD

Marie Prot-Taillandier1, Carlos Afonso1, Sverine Zirah2, Quentin Enjalbert3, Rodolphe Antoine3, Jrme Lemoine3, Philippe Dugourd3, Sylvie Rebuffat2, Jean-Claude Tabet1
1 2
Institut Parisien de Chimie Molculaire, UMR 7201 CNRS-UPMC, 4 place Jussieu , 75005 Paris, France
Molcules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS-MNHN, 57 rue Cuvier, 75005 Paris, France
3
Laboratoire de Spectromtrie Ionique et Molculaire, UMR 5579 CNRS-UCBL, 43, bd du 11 Novembre 1918, 69100 Villeurbanne, France Contact: Marie Prot-Taillandier (marie.perot@u-psud.fr)
Keywords: Systems biology Les peptides lasso sont des peptides bioactifs dorigine bactrienne. Lextrmit C-terminale de ces peptides est enchsse dans un cycle macrolactame form entre le rsidu N-terminal et le carboxylate de la chane latrale d'un rsidu glutamate ou aspartate en position 8 ou 9. La maturation dun prcurseur linaire sous laction de deux enzymes permet dobtenir cette structure compacte et rigide. Notre travail consiste caractriser par spectromtrie de masse la topologie de ces peptides en forme de lasso. Leur fragmentation est compare celle de peptides synthtiques possdant une topologie diffrente avec le mme enchanement dacides amins: peptide lactame non-lasso ou peptide linaire. La microcine J25 (MccJ25), peptide lasso antibactrien produit par Escherichia coli AY25 [1], a dabord t tudie par spectromtrie de masse en mode positif (CID, IRMPD et ECD) [2]. Dans cet expos nous prsenterons les rsultats obtenus sur cette molcule en mode ngatif par activation vibrationnelle (CID) et lectronique (EDD, EPD). Les expriences EPD (Electron Photodetachment Dissociation) ont t ralises sur un LTQ modifi quip dun laser 266 nm [3], tandis que les expriences EDD (Electron Detachment Dissociation) ont t ralises sur un FTICR [2] avec des lectrons denviron 20 eV. La MccJ25 contient deux tyrosines capables dabsorber lUV 266 nm, induisant le dtachement dlectron lorigine des fragmentations observes en EPD. Afin de prciser le rle de chaque tyrosine en EPD, nous avons tudi deux variants de MccJ25 produits par mutagense dirige, dans lesquels chaque tyrosine est slectivement substitue par une phnylalanine. Par ailleurs des expriences de double-rsonance ont t ralises en EDD sur le FT-ICR en faisant varier lamplitude djection de lion de charge rduite au cours de lirradiation par les lectrons [4]. Ces expriences fournissent des informations sur la cintique de formation des fragments et nous ont permis de diffrencier plusieurs mcanismes de fragmentation. [1] Rosengren K.J et al, Biochemistry 43, 4696 (2004) [2] Zirah S et al, J. Am. Soc. Mass Spectrom. 22, 467 (2011) [3] Larraillet V et al, Anal. Chem. 81, 8410 (2009) [4] Lin C et al, J. Am. Soc. Mass Spectrom. 17, 1605 (2006) Link: http://hal.archives-ouvertes.fr/docs/00/57/84/42/PDF/Article.pdf
280/381
[P194] Lipid Imaging by Liquid Extraction Surface Analyses of brain tissue sections using High Resolution Nano-ESI-MS and Nano-LC-MS.
Reinaldo Almeida1, Johannes Vogt3, Hans Kristian Hannibal-Bach2, Jan Baumgart3, Robert Nitsch3, Christer Ejsing2, Kees Vlak1
1 2 3
ADVION, Harlow Enterprise Hub, CM20 2NQ Harlow, Essex, UK Department of Biochemistry and Molecular Biology, , Odense, Denmark Institute For Microscopical Anatomy and Neurobiology, , Mainz, Germany
Contact: Kees Vlak (kvlak@advion.com) Keywords: Imaging mass spectrometry, Instrumentation Novel Aspect: Comprehensive Brain Tissue Lipid Imaging by Liquid Extraction Surface Analyses (LESA) in combination with nano-ESI and nano-LC-MS, which enables an extra separation step after the tissue extraction . Introduction Liquid Extraction Surface Analyses (LESA) can have distinguished advantages providing soft ionisation of analytes without introducing additional sample preparation steps like matrix deposition. Although the spatial resolution of LESA ( ~ 200 m) is lower than that of MALDI ( 10 -100m), LESA allows the sensitive detection of a wide range of lipids classes in combination with Nano-ESI-MS and Nano-LCMS. Methods Frozen Mice brain tissue, horizontally sliced in 15 m thickness and placed onto a glass slide, was analyzed by liquid extraction surface analyses. 0.5 L to 1 L optimized extraction solvent was robotically placed onto the tissue to form a micro liquid junction between a capillary and the surface. The extract was then aspirated into the capillary and transferred automatically to the inlet of a nano ESI chip for direct analyses in a shotgun lipidomics fashion using high resolution mass spectrometry detection. Alternatively the extract was loaded onto a nanobore column enabling the chromatography separation and localization of individual Lipid species in single MS analyses. In booth modes positive and negative Ion detection was performed to increase the coverage. Preliminary Data A crucial aspect for a successful analysis is to control the extraction of a defined area on the biological surface of interest. The surface tension of the solvent and the capillary forces had to be optimized for best extraction and ionization efficiency maintaining the highest possible spatial resolution. A mixture of 1/5 Chloroform/Methanol with 5mM Ammonium Acetate for positive mode analyses and 0.005% Methylamine for negative mode worked best. Furthermore the transfer capillary placing the solvent onto the tissue was optimized to have 100um ID and 170 um OD resulting in a 200um spatial resolution for this technique. Using Lipid imaging of specific regions of mice brain hippocampus like the dentate gyrus, the CA1 region or the layers 2/3 of the entorhinal cortex, we identified, quantified and localized several Lipid species including PC,PE,PA,PS,PI,SM, Sulfatide, Cholesterol, FFA and LPA. 1 uL extract allowed the analyses for 20 minutes at a flow rate of 50 nL/min by direct Infusion using the TriVersa Nanomate (Advion BioSciences) coupled to high resolution MS (LTQ Orbitrap, Thermo Fischer) or by MPIS using the QSTAR (ABSciex). In a second experiment 0.5L 1 L of the extract was automatically loaded onto a PVA-SIL nanobore column (YMC) to furthermore separate isobaric compounds in the Lipid extract. In the hippocampus all pyramidal cells express PRG-1 a membrane Protein interacting with lipid phosphate phosphatase (LPP) allowing the enzymatic dephosphorylation of bioactive PA, LPA and S1P. These lipid phosphates play an important role in signaling and are therefore key in cellular processes. We here compare the Lipid composition in the different hippocampus regions of wild type mice and a PRG-1-KO mice using the LESA shotgun and LESA LC-MS approach in positive and negative ionization mode resulting in the most comprehensive and sensitive Lipid imaging method. Link: www.advion.com
281/381
[P195] Proteomics of CAP-Gly proteins-EB1 C-terminal acidic tail interactome.

David Calligaris 1, Marlne Marcellin 2, Claude Villard 1, Lionel Terras 3, Michel O. Steinmetz 4, Bernard Monsarrat 2, Diane Braguer 1, Pascal Verdier-Pinard 1, Daniel Lafitte1
1
INSERM UMR 911, Centre de Recherche en Oncologie biologique et Oncopharmacologie; Plateforme Proteomique et Innovation Technologique Timone, 27 boulevard Jean Moulin, 13385 Marseille Cedex 5
2 3 4
Institut de Pharmacologie et de Biologie Structurale , 205 route de Narbonne, 31077 Toulouse Synprosis, 59, avenue sainte victoire Zac Saint Charles, 13710 Fuveau Biomolecular Research, Structural biology, Paul Scherrer Institut, OFLC/111, CH- 5232 Villigen
Contact: David Calligaris (callidav@yahoo.fr)
Keywords: Proteins, peptides and small molecules quantification , Signaling, interactomics and post-translational modifications Microtubule +TIPs constitute a dynamic interaction network where the C-terminal motif EEY/F, common to the +TIPs EB1, CLIP-170 and to -tubulin binds to the CAP-Gly domains of certain +TIPs such as p150glued (dynactin1) or CLIP-170. This network in involved in cell motility for instance, but the nature and the changes in +TIPs-containing complexes under the influence of different extracellular cues and over time are still to be determined. Towards this goal, we use EB1 C-terminal peptides coupled to beads to isolate CAP-Gly proteins containing complexes from total cell lysates, prior to analysis by mass spectrometry. We synthesized peptidic probes corresponding to the extreme C-terminus of EB1, containing or not the C-terminal tyrosine residue necessary for interaction with CAP-Gly domains, and a cysteine added at the N-terminus to immobilize them on bead surface. Linear and reflectron mode MALDI mass spectrometry analyses of these probes showed a mass spectrum characteristic of each peptide. A major product ion due to a preferential fragmentation at proline N-terminus was present in both spectrums. Therefore, by using direct MALDI-TOF analysis of recombinant CAP-Gly domain binding to these immobilized peptides we observed both the peptide linkage on beads and the specific binding of recombinant CAP-Gly domains on tyrosinated probes. This validation of our peptidic probes warranted their use for the isolation of CAP-Gly proteins containing complexes from total cell lysates. We obtained a first proof of principle showing by Western blotting the specific binding of CLIP-170 from HMEC-1 endothelial cells on tyrosinated EB1 probes. Quantitative mass spectrometry based analysis of revealed proteins associating specifically to EB1 last 15 residues. We will use these tools in different cell models and for the search of inhibitors of this peculiar mode of protein-protein interaction.
282/381
[P196] Analyse shotgun du scrtome de cellules pithliales pour des applications en toxicologie
Carine Darolles1, Laetitia Chardan1, Stamatiki Roussi1, Jean Charles Gaillard1, Nicole Sage1, Jean Armengaud1, Vronique Malard1
1
Laboratoire de Biochimie des Systmes Perturbs, SBTN, iBEB, DSV, CEA, BP17171, BAGNOLS SUR CEZE CEDEX 30207 Contact: Vronique Malard (veronique.malard@cea.fr)
Keywords: Systems biology, Clinical proteomics Le terme scrtome regroupe lensemble des protines libres par un systme biologique dans des conditions donnes. La scrtion des protines est finement rgule et reflte ltat cellulaire. Les approches les plus rcentes en protomique offrent la possibilit didentifier la majorit des protines prsentes dans un chantillon biologique mais les difficults techniques rencontres sont relles: grer la dilution des protines scrtes dans les grands volumes de fluides biologiques ou de milieu de culture, discriminer les protines scrtes des protines cytoplasmiques ventuellement relargues en cas de mort cellulaire, et enfin viter ou du moins limiter, le masquage des protines scrtes par celles initialement prsentes dans le fluide biologique ou le milieu de culture, notamment les protines du srum. Nous tudions le scrtome des cellules BEAS-2B, une ligne de cellules pulmonaires humaines immortalises, en rponse une intoxication au cobalt, mtal toxique induisant diverses pathologies respiratoires et cutanes lors dexpositions professionnelles. Une phase doptimisation a consist tester plusieurs milieux de culture appropris. Pour chaque milieu slectionn, ladaptabilit des cellules a t vrifie en comparaison avec le milieu recommand via lestimation de la prolifration, de la survie, et des observations de profils de cycle cellulaire. Nous avons ensuite analys en spectromtrie de masse en tandem des chantillons de scrtome obtenus en absence de cobalt. Suite ces premires tapes, les chantillons de scrtomes obtenus en prsence de cobalt une dose faiblement toxique ont t gnrs dans le milieu optimal pour lanalyse shotgun. Nous avons pu identifier 21 protines scrtes signant la toxicit du cobalt. Ces protines reprsentent des pistes intressantes de recherche en toxicologie.
283/381
[P197] Autoantibody profiling using immunoproteomic analysis of sera of patients with a new type of autoimmune-like hepatitis occurring after bone marrow transplantation.
Elvire Beleoken1, Rodolphe Sobesky1, Jean-Pierre Le Caer3, Jean-Henry Bourhis4, Mylne Sebagh1, Catherine Guettier1, Catherine Johanet1, Didier Samuel1, Eric Ballot1, Jean-Charles Duclos-Valle1
1 2 3
INSERM U785, Hpital Paul Brousse, 14 Avenue Paul Vaillant Couturier, 94800 Villejuif, France Universit Paris-Sud, Facult de Mdecine, 63 Avenue Gabriel Pri, 94270 Le Kremlin Bictre, France
CNRS/Institut de Chimie des Substances Naturelles, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
4 5
Institut Gustave Roussy, 39 bis rue Camille Desmloulins, 94800 Villejuif, France Laboratoire d'Immunologie, Hpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012 Paris, France
Contact: Jean-Charles Duclos-Valle (jean-charles.duclos-vallee@pbr.aphp.fr)
Keywords: Clinical proteomics Occurrence of auto-immune-like hepatitis developed de novo is exceptionally reported after bone marrow transplantation (BMT). The purpose of this study is to characterize autoantigens recognized by sera of such patients by using serological proteome analysis. Patients and methods. Three patients (one female, two males) received allogeneic BMT for myelodysplastic syndrom (n=1) or chronic lymphocytic leukaemia (n=2). In the year post-BMT, after stopping the immunosuppressive therapy, they developed an auto-immune-like hepatitis according to the hepatic histological features, different from graft-versus-host disease (GVHD), particularly without bile duct injury. Prothrombin times were between 37 and 100%, aminotransferase levels from 13 to 72 times the upper normal limit and bilirubin from 82 to 270 mol/L. Serology of viral hepatitis was negative. Sera before and during the autoimmune hepatitis (AIH) occurrence were tested on immunoblots performed with two-dimensional electrophoresis resolved proteins of cytosolic, microsomal, mitochondrial and nuclear fractions obtained from rat liver homogenate. Antigenic targets were identified by mass spectrometry. Results. Immunoreactive spots stained by sera contemporaneous with AIH were more numerous and more intensely expressed than those stained by sera taken before the hepatic disorder for all cellular fractions used as antigen. A great pattern heterogeneity depending on the patient was also noted and a total of 259 spots were stained exclusively by sera taken at the moment of hepatic disorder. 227 spots were actually identified, corresponding to 110 different proteins, some spots being isoforms of the same protein. Thirteen of them were common targets of antibodies present in all patient sera and often found in auto-immune disorders. Conclusion. This is the first immunological description of a new type of de novo-AIH occurring after BMT. The thirteen common antigenic targets differ from those reported in de novo-AIH occurring after hepatic transplantation. The existence of an hepatic variant of GVHD can be put under consideration.
284/381
[P198] Two Dimensional SEC / RP Capillary LC for Top-down Proteomics Analysis

Claude Netter1, Robert van Ling2, Evert-Jan Sneekes3, Marco Karsten3, Wim Decrop3, Remco Swart3
1 2 3
Thermo Fisher Scientific, 26, avenue Duguay-Trouin, 78960 Voisins le Bretonneux, France Thermo Fisher Scientific, , Olten, Switzerland Thermo Fisher Scientific, , Amsterdam, The Netherlands
Contact: Claude Netter (claude.netter@thermofisher.com)
Keywords: Separative analysis methods Top-down proteomic LC-MS aims at investigating protein structure and post-translational modifications (PTM) through liquid phase separation and MS analysis of intact proteins. The advantage of top-down approaches over bottom-up approaches is the absence of a proteolytic cleavage step. This keeps all protein information within one molecule and does not multiply sample complexity. However, working with intact proteins introduces challenges for the analytical technology used. Proteins are more difficult to separate than peptides using standard LC methods and more difficult to study by MS. Recently, different electron based dissociation techniques, such as ECD and ETD have been successfully applied to intact proteins to obtain sequence and PTM information. The successful application of these techniques in tandem mass spectrometry for protein mixtures requires a separation step but powerful liquid chromatographic methods for proteins are scarcely published and need to be developed and optimized for direct interfacing with mass spectrometry. In this study we have developed a two dimensional capillary scale LC method for the separation of intact proteins. Proteins are separated and fractionated on a capillary size exclusion column. Next, proteins contained in the size fractions are separated on reversed phase capillary monolithic columns prior to UV and MS detection. The method has been optimized with respect to SEC fractionation and RP gradient conditions for standard proteins and complex mixtures.
285/381
[P199] Optimizing Particle Size and Column Length, what is the best way to utilize nano UHPLC in proteomics?
Claude Netter1, Robert van Ling2, Evert-Jan Sneekes3, Karsten Dekker3, Bjorn de Haan3, Remco Swart3
1 2 3
Thermo Fisher Scientific, 26, avenue Duguay-Trouin, 78960 Voisins le Bretonneux Thermo Fisher Scientific, , Olten, Switzerland Thermo Fisher Scientific, , Amsterdam, The Netherlands
Contact: Claude Netter (claude.netter@thermofisher.com)
Keywords: Separative analysis methods The development of UHPLC capabilities on nano LC scale has opened up new ways to provide proteomics researchers with the peak capacity they need for their work. UHPLC principles involve the use of smaller particles allowing faster analysis and/or better resolution; it is usually focused on throughput in small molecule analysis. However in proteomics analysis speed is of lesser concern compared to peak capacity. The increased pressures in UHPLC can be used to support increased column lengths as well. With 25 and even 50 cm columns being implemented in routine applications, the 1 meter barrier has become visible. Here we utilize the full potential of nano UHPLC to determine the effects of smaller particles and column length on protein identification. In this study we used different nano columns at the highest possible pressure to separate a complex tryptic digest. The particle size and the length of the column were varied to create columns that operate at upper end of the system pressure limits. All columns were operated at typical nano flowrates of 200-300 nl/min and directly interfaced (ESI) with a mass spectrometer. The gradient length was varied to determine the optimal gradient for each column length. Peak capacity, sequence coverage and protein identifications were used to determine the efficiency of the separation. A smaller particle, often associated with UHPLC, will generate more backpressure. Therefore a 50 cm long column packed with 2 m particles already operates at 750-800 bars, which is the upper pressure range of the used nano LC instrument. Increasing the particle size to 3 m allowed column lengths of 1 meter and operation in the same pressure range. Higher temperatures and smaller particles allow even longer columns. When using these extremely long columns, gradients have to be optimized. Running a too steep gradient will not use the full potential of the column. Only when the gradient length is shallow enough the full length of the column could be utilized and peak capacities approaching 1000 were reached.
286/381
[P200] TFA ion suppression in LC ESI-MS/MS? Beating the dogma with crushing evidences using nano rapid chromatography.
Virginie Salnot1, Laila Sago1, Patrick Mayeux1, Franois Guillonneau1
1 2 3
Plateforme protomique Universit Paris Descartes 3P5, 22 rue Mchain, 75014 Paris INSERM u1016, 22 rue mchain, 75014 Paris CNRS UMR 8104, 22 rue mchain, 75014 Paris
Contact: Franois Guillonneau (francois.guillonneau@parisdescartes.fr)
Keywords: Separative analysis methods, Instrumentation, Proteins, peptides and small molecules quantification Ion suppression is common knowledge when Trifluoroacetic acid (TFA) is used in LC solvents for ElectroSpray Ionisation-MS/MS. However it should also be common knowledge that TFA acts as a much better counter ion for chromatographic resolution in reverse phase LC due to its hydrophobic properties. We show here that both statements are true; however when stationary phases and chromatographers improve concomitantly, TFA-related ion suppression cease to be a pain in the MS, while Formic acid (FA) remains limited to low abundance and low complexity samples. In this work we present a results comparison from similar samples of various concentrations, analysed either with TFA or FA as counter ions in LC solvents. We show evidences of how greatly peptide retention and LC resolution improve with TFA especially when dealing with abundant (concentrated) and complex samples. Link: http://3p5.medecine.univ-paris5.fr/
287/381
[P201] Targeted Protein Quantification in Clinical Samples Using a New Quadrupole-Orbitrap Mass Spectrometer
Elodie Duriez1, Sbastien Gallien1, Nina Khristenko 1, Bruno Domon1, Zhiqi Hao2, Markus Kellmann3, Thomas Moehring3, Andreas Huhmer2
1 2 3
Luxembourg Clinical Proteomics Center, 1A rue Thomas Edison, 1445 Strassen, Luxembourg ThermoFisher Scientific San Jose, 355 River Oaks Parkway, 95134 San Jose, California ThermoFisher Scientific Bremen, Hanna-Kunath-Strae 11, 28199 Bremen, Germany
Contact: Elodie Duriez (elodie.duriez@crp-sante.lu) Keywords: Clinical proteomics Introduction For clinical studies, targeted protein quantification is routinely performed on triple quadrupole instruments. In this study, the performance of a novel hybrid mass spectrometer coupling a quadrupole to an orbitrap mass analyzer was evaluated as an alternative to a classical triple quadrupole approach for the evaluation of biomarker candidates in clinical samples. This new hybrid quadrupole / orbitrap instrument offers true high mass accuracy and high resolution capabilities. Methods Analyses were performed on clinical samples from healthy donors. Sample preparation consisted in protein precipitation, trypsin proteolysis, and desalting on C18 reverse phase cartridges. Analysis were performed on a nano-HPLC system (Dionex, Netherlands) coupled with a new hybrid quadrupole/orbitrap instrument (QExactive, ThermoFisher Scientific). Targeted peptides were selected by the quadrupole and eventually transferred into the orbitrap mass analyzer for quantitative analysis in full scan (SIM) mode. Quantitative experiments involving the MS/MS mode were conducted on several different precursor ions fragmented simultaneously. Quantification was carried out post-acquisition using the elution traces of specific fragment ions. For a study of performance comparison, all the experiments were replicated on a triple quadrupole instrument (TSQ Vantage, ThermoFisher Scientific) operated with intelligent-selected reaction monitoring (iSRM) mode. Results The different modes of operation of the new hybrid quadrupole / orbitrap instrument were applied to the targeted analysis of proteins, i.e., in full scan (SIM) mode and in MS/MS mode. In contrast to triple quadrupole experiments, the quantification of the fragment ions is performed post-run and thus no a priori knowledge of MS/MS fragmentation pattern is required to design acquisition method. To establish the analytical performance, dilution series of 30 isotopically labeled reference peptides corresponding to three exogenous yeast proteins and ten endogenous human proteins were performed to determine the limits of detection and quantification, and the linear dynamic range of the method. Preliminary results have shown limits of detection below 100 amol. To evaluate the performance of the instrument in complex biological matrices, the reference peptides were spiked in the digested clinical samples to perform dilution curves. Based on existing calibration curves, the amount of corresponding endogenous protein in clinical samples was precisely determined. Conclusion This new hybrid quadrupole / orbitrap instrument has significant advantages for clinical application, especially for the unambiguous detection and precise quantification of a large set of candidates in one LC-MS/MS run.
288/381
[P202] MzDB: an optimized file format for the efficient analysis of LC-MS datasets
David Bouyssi1, Matthew Chambers2, Sara Nasso3, Odile Burlet-Schiltz1, Bernard Monsarrat1
1
Laboratoire de protomique et spectromtrie de masse des biomolcules, IPBS, Universit de Toulouse, CNRS, 205 route de Narbonne, 31077 Toulouse
2
Department of Biomedical Informatics, Vanderbilt University Medical Center, 400 Eskind Biomedical Library, 2209 Garland Ave Nashville, USA
3
Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich
Contact: David Bouyssi (david.bouyssie@ipbs.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics, Proteins, peptides and small molecules quantification To overcome issues related to raw data file access the proteomics community produced data file standards such as mzXML [1] or mzML [2]. Although these new formats simplified data sharing among different laboratories, some authors demonstrated [3,4,5,6] that they were not the most efficient solution for high-throughput data processing, such as MS signal extraction in label-free quantitative methods. In order to speed-up data access, indexing strategies have emerged [6] but have not yet been developed into a portable file format. We propose here a new file format, called mzDB, which is a SQLite file with an optimized index for LC-MS 2D range queries. Data can be easily accessed from any programming language with SQLite bindings (all major languages). The main concept of this work relies on the cutting of MS spectra into slices of a given m/z window. Scan slices belonging to the same m/z window are grouped into an entity called run slice. Indexing data through the run slice structure allows performing fast queries in the m/z and time dimensions. More than 30000 MS peptide ion features were extracted in less than 5min from a raw file corresponding to a complex protein mixture (120min nanoLC-MS acquisition). We developed a C++ program which is able to convert vendor raw files into the mzDB format. To avoid the development of manufacturer specific code we used the C++ ProteoWizard framework [7] which handles the main raw data formats. The mzDB format enables the fast processing of a large number of LC-MS raw files which is a critical point for the analysis of emerging proteomic clinical studies. 1: Pedrioli PG et al., Nat Biotechnol. 2004 Nov;22(11):1459-66. 2: Martens L et al., Mol Cell Proteomics. 2011 Jan;10(1):R110.000133. 3: Lin SM et al., Expert Rev Proteomics. 2005 Dec;2(6):839-45. 4: Askenazi M et al., Nat Methods. 2009 Apr;6(4):240-1. 5: Shah AR et al., J Am Soc Mass Spectrom. 2010 Oct;21(10):1784-8. 6: Nasso S et al., J Proteomics. 2010 Apr 18;73(6):1176-82. 7: Kessner D et al., Bioinformatics. 2008 Nov 1;24(21):2534-6.
289/381
[P203] Spectromtrie de masse et sources dionisation atmosphrique : une nouvelle manire daborde le couplage chromatographique et lanalyse MS 3D.
Hugues Preud'homme1, Jean-Marc Joumier2, Ryszard Lobinski1
1 2
Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, 2 avenue Angot, 64053 PAU WATERS S.A.S., BP 608, 78056 Saint-Quentin en Yvelines cedex
Contact: Hugues Preud'homme (hugues.preudhomme@univ-pau.fr)
Keywords: Organic and inorganic mass spectrometry, Separative analysis methods, Instrumentation Afin de rpondre aux besoins actuels en nergie dans le monde, lun des grands dfis est de tenter de mieux caractriser les sources dhydrocarbures non conventionnelles, damliorer leurs taux de rcupration et daugmenter la rentabilit des champs matures (Khan et al., 2006). De nombreux procds capables de favoriser et daugmenter cette rcupration sont labors depuis plusieurs annes. Cependant, ce nest que grce la description prcise dun gisement (communications inter-puits, htrognits, permabilits, estimation de la quantit dhuile rsiduelle saturation (SOR), etc.) quil est possible de dfinir et doptimiser les mcanismes de rcupration assiste (injection deau, de gaz, de polymres, etc.). La surveillance de molcules ou de contaminants chimiques ltat de traces reprsente de nos jours, de vastes applications dans le domaine des sciences analytiques, de lenvironnement, le dveloppement et lutilisation raisonne de nos ressources. Ces techniques, notamment base sur les sources dionisation pression atmosphrique, sont dsormais reconnues comme loutil dinvestigation de choix pouvant sappliquer aussi au suivi de la qualit des eaux souterraines et de surface. Lutilisation de mthodes de chromatographies rapides en phase gazeuse ou liquide couples des analyseurs de masse utilisant ces sources permettent denvisager le protocole danalyse sous un autre angle en minimisant le temps de prparation, voire quelque fois en privilgiant lanalyse directe tout en gardant des performances similaires avec une LOQ< 1ng/g. LUHPLC/MS-MS pour lanalyse directe de traceurs aromatiques halogns ou organomtalliques peut tre ralise en moins de 5 minutes pour des eaux de gisement prsentant des taux de salinits allant de 5g/l 130g/l. Applique lanalyse dchantillons rels issus de campagnes de traage en cours, cette approche analytique originale a permis de remplacer avantageusement la technique de rfrence base sur une extraction drivation prconcentration (1h30) avant analyse par GC/MS ou GC/MSMS (1h) pour des performances similaires. Une approche similaire peut tre employe pour lanalyse dorganomtalliques en milieu aqueux ou la technique de rfrence est la GC-ICPMS. Lapparition dune source pression atmosphrique pour chromatographie en phase gazeuse sur les spectromtres de masse de dernire gnration permet de raliser une approche originale an associant les 3 sources pression atmosphrique (ESI, ICP et APGC) en travaillant avec des conditions chromatographiques identiques et sans limitation en raison de lutilisation dune source pression rduite. Cela facilite grandement lalignement des chromatogrammes et permet deffectuer de confirmer une structure dun inconnu (ESI/ APGC - MS) avec la spcificit du suivi dlments traces et dune quantification sans standards molculaires (ICP - MS). Link: www.lcabie.com
290/381
[P204] Comparative analyses of protein expression of strain UBSA 355 - Tistlia consotensis, a new species isolated from a terrestrial saline spring
Carolina Rubiano-Labrador1, Sandra Baena1, Jean Armengaud2
1
Unidad de Saneamiento y Biotecnologa Ambiental, Departamento de Biologa, Pontificia Universidad Javeriana, Carrera 7 n40-62, POB 56710, 110121 Bogot, Colombia
2
Laboratoire de Biochimie des Systmes Perturbs, CEA Marcoule, DSV, iBEB, SBTN, LBSP, BP17171, 30207 BAGNOLS-SUR-CEZE, France Contact: Sandra Baena (baenasandra2004@yahoo.es)
Keywords: Systems biology, Proteins, peptides and small molecules quantification , Separative analysis methods The objective of this study is to analyze qualitatively and quantitatively the expression profile of extra and intracellular proteins of strain UBSA 355 - Tistlia consotensis, under different culture conditions. Tistlia consotensis, is a moderate halophilic microorganism identified as a new species of subclass -proteobacteria, distantly related to genus Thalassobaculum (90% 16S rRNA gene sequence similarity). The proteomic approach contributes to the knowledge on its metabolic pathways, enzymatic potential and its mechanisms of adaptation to specific conditions of growth. We will evaluate different salinity conditions for growth: 0, 0.5 and 4% NaCl (w/v), which correspond to minimal, optimal and maximum concentrations supported by this strain, respectively. In order to achieve greater coverage of the proteome different proteomic strategies are currently under investigation: two-dimensional electrophoresis (2-DE), multidimensional chromatography coupled to tandem mass spectrometry (Mud-PIT) and 1-D gel electrophoresis followed by nanoLC-MS/MS. As the genome of this microorganism is not yet sequenced, novel approaches to interpret proteomic data are carried out. The proteomic profiles from cells grown in different culture conditions should lead to novel insights into the biological responses related with adaptive mechanisms to salinity stress.
291/381
[P205] Le couplage Mobilit Ionique - Spectromtrie de Masse (IM-MS) pour le suivi de changements conformationnels induits par la fixation dun ligand sur une cible protique.
Stphanie Petiot1, Jean-Paul Renaud2, Denis Zeyer2, Valrie Vivat Hannah2, Alain Van Dorsselaer1, Cedric Atmanene2, Sarah Sanglier-Cianfrani1
1
Laboratoire de Spectromtrie de Masse Bio-Organique (LSMBO), IPHC, Universit de Strasbourg, CNRS UMR7178, ECPM - Bat. R5.0 - 25 rue Becquerel, 67087 Strasbourg
2
NovAliX, Structural Biology, Bioparc, Bd Sbastien Brant, BP30170, 67405 Illkirch
Contact: Alain Van Dorsselaer (vandors@unistra.fr)
Keywords: Ionic mobility Le couplage Mobilit Ionique / Spectromtrie de Masse (IM-MS) est technique analytique mergente permettant dapporter des informations structurales sur la taille et la forme dun complexe supramolculaire, par le biais de mesures de sections efficaces (CCS). Alors que de nombreux articles utilisent le couplage IM-MS pour suivre des changements conformationnels induits par la fixation de protines sur des complexes multi-protiques de hauts poids molculaires [1,2], peu/pas de donnes sont disponibles dans la littrature sur les possibilits du couplage IM-MS pour le suivi de changements conformationnels induits par la fixation dune petite molcule (ligand) sur une protine. Or, dans bien des systmes biologiques, des changements structuraux fins sont lorigine dactivits physiologiques importantes. Des travaux antrieurs nous ont permis de montrer dans le cas de complexes de plusieurs centaines de kDa que des changements de conformation conduisant une modification de lordre de 5% de la CCS pouvaient tre mis en vidence avec les instruments commerciaux IM-MS de premire gnration (Synapt HDMS G1, Waters) [3]. Lobjectif de ce travail est de montrer que le couplage IM-MS constitue une technique analytique de choix pour dtecter des changements conformationnels trs fins dans le cas de systmes protine/ligand. Nous insistons ici sur limportance de loptimisation instrumentale pour lobtention dune rsolution optimale en mobilit ionique. Limportance de ces paramtres instrumentaux sera illustre au travers de lexemple du systme protine/ligand Bcl-Xl/ABT-737. La protine Bcl-Xl (B-cell lymphoma-extra large) est implique dans de nombreux cancers du fait de son caractre anti-apoptotique favorisant la prolifration cellulaire [4]. La molcule ABT-737 a t dcrite comme capable dactiver lapoptose en se liant Bcl-Xl avec une forte affinit (Ki 1 nM) [5]. Nos premiers rsultats montrent que, moyennant une optimisation des paramtres instrumentaux prcise et rigoureuse, le couplage IM-MS permet dsormais de dtecter des changements conformationnels subtils. [1] Ruotolo BT et al., Nat. Protoc., (2008), 3, 1139-1152. |2] Atmanene C et al., Anal. Chem., (2009), 81, 6364-6373. [3] Atmanene C et al., Anal. Chem. (2010), 82, 3597-3605. [4] Witham J. Clin. Cancer. Res. (2007), 13, 7191-7198. [5] Oltersdorf T et al., Nature. (2005), 435, 677-681.
292/381
[P206] Coupling of a linear ion trap with a VUV beamline

Alexandre Giuliani1, Francis Canon1, Matthieu Refregiers 1, Laurent Nahon1
1 2
Synchrotron SOLEIL, L'Orme des Merisiers, 91129 Gif-sur-Yvette INRA, U1008 CEPIA, Rue de la Graudire, 44316 Nantes
Contact: Alexandre Giuliani (alexandre.giuliani@synchrotron-soleil.fr)
Keywords: Instrumentation A novel experimental system for tandem mass spectrometry and ion spectroscopy of electrosprayed ions in a linear quadrupole ion trap using VUV synchrotron radiation is presented. The classical activation methods of the precursor ion in tandem mass spectrometry are usually performed via collisions with a gas. However, photon activation can offer a possibility to selectively release large amount of internal energy into the ions of interest, allowing performing spectroscopy on large isolated biological species, as well as on small ligand molecules forming non-covalent complexes. Trapped ions spectroscopy, however, is a rather challenging task because of the limited ion densities, as well as limited available photon fluxes. The synchrotron radiation MS/MS method implies the following sequence of events: 1) electrosprayed ions are injected, selected and stored in the trap; 2) when the desired ion capacity is reached, the beam shutter opens, thus starting the irradiation; 3) after a desired time of irradiation, the shutter intercept the beam; 4) the mass spectrum is recorded; 5) the monochromator is set to the next wavelength and the procedure is repeated. The present experimental setup for spectroscopy of electrosprayed ions was coupled with the DESIRS beamline at the SOLEIL synchrotron radiation facility. The undulator emission allowed measurements in the 4-20 eV photon energy range. The photon beam can be filtered out for high harmonics using a gas filter. The experimental system was based upon a commercial linear quadrupole ion trap (Thermo scientific LTQ XL), equipped with the ESI probe. Calculations were performed to conceive an optimal overlap between the beam and the trapping region, regarding the size and divergence of the light beam, as a function of the photon energy and the trap distance from the focal point. The synchrotron beam is introduced into the trap through the back lens of the spectrometer. A special frame has been constructed to allow fine-tuning of the position of the spectrometer, ie of the trapping region, with respect to the light beam. The vacuum manifold with a turbo pumping stage has been designed to accommodate pressure difference between the beamline (10-8 mbar) and LTQ (10-5 mbar). The manifold also includes elements for additional filtering of high order radiation, photon flux measurements and photon beam shutter. The shutter has been designed to achieve short (5-10 ms) and reproducible chopping time under high-vacuum conditions. The most recent obtained results include, for the first time on biopolymers, the photodetachment spectroscopy using first-order synchrotron light of peptide polyanions and the first ionization spectroscopy of polycations. Beside the fundamental interest of accessing physical properties of large biological ions isolated in vacuo, the present work could lead to a new, efficient and powerful MS/MS technique.
293/381
[P207] Analyse des rponses hpatiques de lorganisme dnutri : approches protomiques/transcriptomiques

Thierry Wasselin1, Alain Van Dorsselaer1, Fabrice Bertile1
1
Institut Pluridisciplinaire Hubert Curien, LSMBO, CNRS-UdS, 25 rue Becquerel, 67087 Strasbourg
Contact: Fabrice Bertile (fbertile@unistra.fr)
Keywords: Systems biology, Clinical proteomics Contexte La malnutrition et la dnutrition sont au aujourdhui au rang des menaces les plus grave pour la sant publique. Pourtant le traitement efficace des patients svrement dnutris souffre encore dun manque de connaissance des rponses physiologiques la privation de nourriture longterme. Le foie est essentiel au maintien de lhomostasie nergtique, la synthse de multiples protines, la rgulation du mtabolisme de certaines hormones Il est lun des tissus les plus affects lors de transitions nutritionnelles. Les fonctions hpatiques sont donc probablement altres dans de telles situations, risquant de compromettre lintgrit de lorganisme sur le long-terme. Objectifs Lobjectif de cette tude tait danalyser les effets du jene prolong sur le protome et le transcriptome hpatiques chez le Rat. Les donnes ont t analyses en relation avec les transitions mtaboliques associes la rponse au jene, en comparant la phase 2 (mobilisation des substrats lipidiques) et la phase 3 (dgradation des protines corporelles) ltat nourri. Mthodes Les analyses protomiques ont combin lectrophorse bidimensionnelle et spectromtrie de masse (MALDI-TOF-MS). Les analyses transcriptomiques ont utilis des puces ADN (Affymetrix). Les donnes omics ont t confirmes par dautres mthodes (Western-blot et Northern-Blot). Des tests fonctionnels ont t raliss pour renforcer et valider la pertinence biologique des donnes omics . La significativit des rponses a t teste par ANOVA et tests de Tukey. Rsultats Les donnes montrent que le jene prolong induit, de faon phase-spcifique, des effets dltres pour plusieurs processus biologiques, incluant les mcanismes de dfenses contre le stress oxydatif et les processus de folding des protines, mais aussi les fonctions mtaboliques. Les tests fonctionnels ont confirm que les dommages oxydatifs sont fortement augments en phase 3, et que le statut nergtique du foie est alors particulirement altr. Conclusions Les rponses adaptatives rapportes par nos donnes permettent de proposer que le foie est incapable de maintenir des fonctions saines lors dun jene long-terme. De telles altrations pourraient tout fait tre ressenties par le systme nerveux central, qui en retour pourrait stimuler le comportement alimentaire (caractristique de la phase 3) afin de promouvoir la survie.
294/381
[P208] Characterization of serum proteins interacting with nanoparticles

Arnaud Lamarca1, Christian L. Villiers3, Rachel Couderc3, Philippe Bulet1, Patrice N. Marche3
1 2
Plateforme Biopark, Btiment le Forum, 74160 Archamps
AGe Imagerie et Modlisation, AGIM/UJF CNRS FRE 3405, Pavillon Taillefer, Facult de medecine de Grenoble, 38706 La Tronche
3
Immunologie analytique des pathologies chroniques, INSERM/UJF U823, Rond point de la chantourne, 38706 La Tronche Contact: Arnaud Lamarca (arnaud.lamarca@agim.eu)
Keywords: Systems biology, Separative analysis methods, Signaling, interactomics and posttranslational modifications Background: The present study has been conducted in line with Cellnanotox (European program). It has already been shown that nanoparticles (NP) can enter the cells, and interfere with the immune system. We previously showed that noncytotoxic, gold NPs significantly modified cytokine secretion (1). During their trafficking through the bloodstream, NPs interact with many plasmatic proteins that may facilitate their interaction with cellular receptors leading to endocytosis or phagocytosis. Methods: NPs were incubated with fresh human plasma. The plasma proteins trapped by the NPs were subjected to trypsin digestion. The digest was analyzed by direct mass fingerprinting (PMF) with MALDI mass spectrometry (MS). To identify and characterize the NPs interacting plasma proteins, the digest was purified by liquid chromatography,and the different peptides were sequenced by tandem mass spectrometry (MS/MS). Data bank (ExPASy, SwissProt) was interrogated under MASCOT. Results: We have investigated the interaction of gold and ferricobalt (FC) NPs with human plasma proteins using direct proteolysis and in gel digestion. For gold NPs we observed that plasma proteins from the complement cascade were attached to the NPs, especially opsonin molecules. Different immunoglobulins were identified; among them some are known to interact with pathogen-internalization receptors. In parallel, we used the same strategy using FC NPs in order to determine the specificity of the interactions. We demonstrated, thanks to the differential PMF, that the composition of the NPs can discriminate between human plasma proteins for the interaction. Conclusion: This study established that NPs, when incubated in fresh human serum, could easily trap circulating proteins involved in processes such as opsonisation and internalization of foreign elements. This suggests that NPs may interact specifically with cells involved in the immune response. Our goal is to demonstrate if coated proteins are important for NPs internalization and for potential ensuing modification of biological activities. References: (1) Villiers, L.C., et al. Analysis of the toxicity of gold nanoparticles in the immune system: effect on dendritic cell functions. J Nanopart Res. 2010. 12:55-60
295/381
[P209] Identification des cibles protiques des voies de la thioredoxine et de la glutathione dans les cellules humaines par une stratgie ocSILAC
Giovanni Chiappetta1, Sega Ndiaye1, Michel Toledano2, Joelle Vinh1
1 2
Spectromtrie de masse biologique et protomique, USR3149, ESPCI, 10 Rue Vauquelin, 75005 Paris LSOC, CEA , Saclay, 91191 Gif-sur-Yvette
Contact: Giovanni Chiappetta (giovanni.chiappetta@espci.fr)
Keywords: Signaling, interactomics and post-translational modifications Introduction. Lidentification des proteinse impliques dans les voies GSH et Trx chez les mammifres est une tape cl pour comprendre les mcanismes molculairesuls par la biochimie rdox des thiols (1). Dans le but de caractriser ltat doxidation des soufres du protome et le profil dexpression protiques dans des cellules HELA dans lesquelles lexpression de la thioredoxine reductase 1(TRR1) ou la GSH est inhibe, nous avons dvelopp une stratgie pour localiser les cystines oxides et, simultanment, gnrer les profils dexpression des protines. Cette stratgie, base sur la technologie SILAC, est nomme ocSILAC. Mthodes. La synthse de GSH est inhibe dans les cellules HELA par exposition 24h la Lbuthionine-[S,R]-sulfoximine. Lexpression de la TRR1 est inhibe par des ARNi. Aprs lyse cellulaire, notre procdure de marquage des soufres impliquant de lacide trichlorioactique est utilise (2). Les soufres rduits sont alkyls avec liodoactamine, et les soufres oxyds sont spcifiquement marqus par le (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide (biotine-HPDP). Rsultats. La stratgie ocSILAC a t conue pour enrichir spcifiquement les peptides contenant les cystines oxydes dans des extraits cellulaires, aprs un marquage SILAC. Pour cela, 1) les cystines oxides sont marques spcifiquement par la biotine-HPDP; puis 2) les analyses LC MSMS des fragments biotinyls sont ralises, permettant de suivre loxidation des soufres dans deux tats cellulaires diffrents. En raison des changements dans les niveaux dexpression protiques, qui peuvent induire des biais, les donnes sont normalises avec le profil dexpression protique en utilisant les analyses LC MSMS de la fraction de peptides libres comme rfrence. La stratgie ocSILAC a t utilise pour tudier les lignes cellulaires dans lesquelles TRR1 et GSH ont t inhibes. En utilisant cette stratgie, nous avons valu ltat rdox de plus de 400 rsidus cystine, et quantifi plus de 300 protines. Conclusion. ocSILAC est une alternative valide la stratgie SILAC pour suivre loxidatin des soufres (3,4). Le point fort decette stratgie est la possibilit de suivre en mme temps les profils dexpression protiques et le niveau de modifications post-traductionnelles. Linterprtation biologique des donnes obtenues dans les lignes cellulaires nexprimant pas GSH et TRR1 est en cours. References 1. Toledano, M.B., et al. (2007). FEBS Lett. 581, 3598607. 2. Delaunay, A., et al. (2000). EMBO J. 19, 5157166. 3. Leichert LI et al. Proc Natl Acad Sci U S A. 2008 105(24):8197-202. 4 Fu C. et al. Mol Cell Proteomics. 2009;8(7):1674-87.
296/381
[P210] EIF3 subunits interaction study by hydrogen/deuterium exchange FT-ICR mass spectrometry
Jianqing Wu1, Pierre-Damien Coureux2, Edith Nicol1, Emmanuelle Schmitt2, Yves Mchulam2, Guillaume van der Rest1
1 2
Laboratoire des Mcanismes Ractionnels, CNRS, UMR7651, Ecole Polytechnique, 91128 Palaiseau, France Laboratoire de Biochimie, CNRS, UMR7654, Ecole Polytechnique, 91128 Palaiseau, France
Contact: Jianqing Wu (wu@dcmr.polytechnique.fr)
Keywords: Systems biology, High mass and high resolution mass Spectrometry Cellular cycle is highly regulated to optimally respond to any extracellular stimuli. Transcriptional control has been well studied to identify the components (proteins, RNA, small molecules) involved in that regulation. However, it seems that the mRNA translation is also very well controlled during gene expression. Moreover, the most regulated step occurs during translation initiation, the rate limiting step in protein biosynthesis. This particular step involves the small subunit of the ribosome and many eukaryotic proteins called translation initiation factors (eIF). Among those, the eIF3 complex plays a crucial role in the architecture of the translation initiation complex and is an important target for drug design. Our studies focused of the eIF3 complex of yeast: made of only 5 subunits, its a simplified form of the human eIF3 complex (made of 13 subunits). Most of the published data of this complex comes from biochemical and cellular studies, but very little is known of the structural organization of the eFI3 complex. We are currently studying the interactions of 3 subunits of the yeast eIF3 complexes: eIF3i (37 kDa), a domain of eIF3b (11.3 kDa) and a domain of eIF3g (16 kDa) using the bottom-up approach of amide hydrogen/deuterium exchange (HX) combined with FT-ICR mass spectrometry (MS). HXMS is a powerful tool in protein structure and interaction studies. According to our liquid chromatographic results, while eIF3bC3 and eIF3gC1C does not interact with each other, they can both form dimer complex with eIF3i, and a trimer complex is formed with the present of the three subunits. In the HXMS approach, the individually synthesized subunits were deuterated and then digested by pepsin, followed by immediate nanoLC separation at 0 C and FT-ICR-MS analysis to reduce the back-exchange to hydrogen. The deuteration levels of specific peptides at different reaction times were obtained by extracting the mass shifts between deuterated and unlabelled peptide, which leaded to a kinetic curve of the amide exchange for this peptide. A higher level of deuteration reflects either an easier accessibility to the solvent, or a less stable secondary structure. In addition, by comparing the deuteration level of peptides from the isolated subunits and the complexes, the interaction sites can be revealed, as a change in accessibility is expected to arise from quaternary structure formation. Adequate sequence coverage was achieved using this set-up of nano-flow liquid chromatography system associated with the high sensibility FT-ICR-MS. In addition, the high mass accuracy of FT-ICR-MS enables direct peptide identification of large-size protein complexes based on exact mass and retention times without further need for systematic tandem MS analysis. These are crucial for this protein structural and interaction study in the absence of any crystallographic information.
297/381
[P211] Structural analysis of complex lipids using MALDI SpiralTOF-TOF with high precursor ion selectivity
Ayumi Kubo1, Yoshiyuki Ito1, Masahiro Hashimoto1, Masaaki Ubukata2, Jun Tamura1, Jyun Onodera1, Akihiko Kusai3
1 2 3
JEOL Ltd., 1-2, Musashino 3-Chome Akishima, 196-8558 Tokyo, Japan JEOL USA, Inc., 11 Dearborn Rd., 01960 Peabody, MA USA JEOL (EUROPE) SAS, Espace Claude Monet - 1, Alle de Giverny, 78290 Croissy-sur-Seine, France
Contact: Akihiko Kusai (kusai@jeol.fr)
Keywords: High mass and high resolution mass Spectrometry, Instrumentation, Systems biology Structural analysis of lipids is often performed with LC-MS/MS such as ion trap mass spectrometer and quadrupole-TOF hybrid tandem mass spectrometer. These tandem mass spectrometers can observe product ion mass spectra produced by the low-energy collision induced dissociation (LE-CID) at the laboratory frame collision energy of below a few hundred electron volts and obtain some structural information of the analytes. Detailed information of the fatty acid moieties in the analyte molecules, however, cannot be obtained. In contrast, highenergy CID (HE-CID) can cleave C-C bonds and provide detailed information of the fatty acids moieties, such as the positions of double-bonds, branching, and hydroxylation by means of charge-remote fragmentation. The MALDI TOF-TOF combined a SpiralTOF [1] and an offset parabolic reflectron for first and second TOFMS, respectively has been developed. The SpiralTOF, which has 17 m flight path, can select a monoisotopic ion so that each fragment pathway is observed as single peak on product ion spectrum. Furthermore, PSD ions are eliminated by electrostatic sectors inside the SpiralTOF. As a result, fragment pathways using HE-CID are preferentially observed on product ion spectrum. In addition, the SpiralTOF-TOF has a high precursor ion selectivity to catch up a monoisotopic ion. In this study, we report structural analysis of lipids by HE-CID using MALDI-SpiralTOF-TOF tandem MS with high precursor ion selectivity. [1] Satoh, T.; Sato, T.; Tamura, J. J. Am. Soc. Mass Spectrom. 2007, 18, 1318. Link: www.jeol.com
298/381
[P212] Combining Top down and Bottom up analyses in a single LCMS experiment
Frank Porbeck1, Reinaldo Almeida2
1 2
Advion BioSciences, Ltd, Koa Hockham Building, CM20 2NQ Harlow,UK
University of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense,Denmark Contact: Frank Porbeck (porbeckf@advion.com)
Keywords: Separative analysis methods, Signaling, interactomics and post-translational modifications, Instrumentation In this work we use bottom up and top down analyses by combining online LCMS with nanoESI infusion for the identification of proteins and characterizing their post-translational modifications during the brewing process. The intact mass measurement and primary sequence determination was performed by online UPLC-MS(MS) with simultaneous fraction collection. The top down affords the post-translational modification observation after the UPLC-MS run by automated nanoESI infusion of the previously collected fractions. Since nanoESI consumes just a small amount of the analyte, proteolytic digestion was performed on the remaining sample for bottom up analyses. This strategy allows us to full characterise proteins involved during brewing, by intact mass determination, PTM characterisation and the corresponding peptide sequence coverage in a single LCMS experiment. The combination of intact protein chromatography by UPLC with a post column splitting system, showed equal separation power and signal to noise ratio, in comparison with the standard non splitting set up. During the online top down analyses the intact mass could be determined by deconvoluting the charge state envelop. However many proteins have been detected but not identified or with low confidence due to the limited time during the elution. These proteins where then targeted during the offline analyses of the previous collected fractions for fop down MS/MS, but also for intact mass determination of very low abundant species. The offline analyses allowed an unlimited averaging capability and optimisation due to the low sample consumption of nanoESI as well as the specific targeting of post translational modifications. Each protein fraction was then digested and reanalysed by offline bottom up, giving again unlimited time for optimising the condition and averaging during the analyses of the corresponding peptides. The good UPLC separation of the intact proteins lead that the peptides identified in each fraction can just correspond to the proteins found by the top down approach previously, adding another confidence level to the analyses. Furthermore the complexity in each fraction is reduced in a way that no 2 dimension chromatography run is needed. The combined top down and bottom up results facilitated the interpretation of unknown proteins as well as post translational modifications. Link: www.advion.com
299/381
[P213] Photoionisation a pression atmospherique de polymeres synthetiques

Vronique Legros1, Bernard Desmazires 2, Alexandre Guiliani3, William Buchmann1
1
Universit d'Evry val dEssonne (LAMBE, UMR 8587), Btiment Maupertuis, Bd. Franois Mitterrand, 91025 Evry, France
2 3
Global Bioenergies, 5 rue Henri Desbruyeres, 91030 Evry, France DISCO beamline, Synchrotron SOLEIL , LOrme des Merisiers, Saint-Aubin, 91192 Gif-sur-Yvette, France
Contact: William Buchmann (william.buchmann@univ-evry.fr)
Keywords: Organic and inorganic mass spectrometry, Separative analysis methods La photoionisation pression atmosphrique (APPI) est une technique dionisation prometteuse pour lanalyse par spectromtrie de masse des polymres synthtiques. Cette technique consiste nbuliser une solution contenant lchantillon analyser, pression atmosphrique, puis ioniser l'chantillon laide de photons mis par une lampe dans lUV. Couple des techniques de sparation liquide, elle peut fournir de nouvelles opportunits pour la caractrisation des polymres, tout spcialement pour les polymres de faible poids molculaire, ou pour les polymres difficilement ionisables par les mthodes dionisation habituelles (MALDI, ESI), typiquement les polymres les moins polaires. En effet, la diffrence de la technique ESI, la technique APPI permet lutilisation de solvants de faible polarit ncessaires pour dissoudre les polymres les plus apolaires. Le processus dionisation en phase gazeuse rend possible lionisation de ces espces, pour lesquels les techniques ESI et MALDI pourraient se rvler inefficaces. Les spectres de masse sont relativement simples interprter, puisque seuls des ions monochargs sont gnrs. Ainsi il ny a pas de superposition possible entre diffrents tats de charge comme cest le cas en ESI. Toutefois, des fragmentations peuvent se produire. Un intrt majeur de lAPPI rside aussi dans sa capacit pouvoir jouer le rle dinterface avec les principales mthodes chromatographiques. La compatibilit avec un plus large panel de solvants quen ESI permet denvisager des couplages, suivants les principaux modes chromatographiques: phase inverse, phase normale, et chromatographie dexclusion strique. Par principe, le couplage LC/MS apporte de nombreux avantages : il permet lidentification des composs dans les pics chromatographiques sans ambigit, limite les effets de suppression de signal et les superpositions entre les espces isobares, problme frquemment rencontr avec les mthodes MS directes. Si la technique APPI semble prometteuse pour lanalyse des polymres, les mcanismes dionisation (ou de fragmentation) ne sont en revanche pas encore bien compris. Pourtant la comprhension de ces mcanismes, est essentielle pour obtenir une estimation fidle des poids molculaires moyens et des indices de polymolcularit. Les sources commerciales APPI utilisent typiquement des lampes Krypton dont lnergie des photons est prdtermine (10eV et 10.6eV). Dans ce contexte, le comportement en APPI de divers polymres standards (polythers, polymthylmtacrylate, polysiloxane, polystyrne) sera abords au travers de quelques exemples. Les mcanismes d'ionisation ont notamment t tudis en utilisant le rayonnement synchrotron SOLEIL qui offre l'opportunit de pouvoir tudier le phnomne dionisation en fonction de l'nergie du photon. Link: http://www.lambe.univ-evry.fr/
300/381
[P214] Liquid chromatography-mass spectrometry assay for the identification and absolute quantification of the apelin peptides in human plasma and bovine colostrum.
Cdric Mesmin1, Franois Fenaille1, Franois Becher1, Eric Ezan2
1
CEA, iBiTec-S, Service de Pharmacologie et dImmunoanalyse, CEA Saclay, Bt 136, SPI/LEMM, 91191 Gif sur Yvette
2
CEA, iBEB, Service de biochimie et toxicologie nuclaire, CEA Marcoule, BP 17171, F-30207 Bagnols-sur-Cze
Contact: Cdric Mesmin (cedric.mesmin@cea.fr)
Keywords: High mass and high resolution mass Spectrometry, Proteins, peptides and small molecules quantification Apelin peptides are of great interest owing to their involvement in physiological and pathological processes and have been proposed as novel biomarkers for heart failure. The main circulating forms have been reported as bearing the 12 (apelin-12), 13 (apelin-13), 17 (apelin17) and 36 (apelin-36) C-terminal amino acids of the 55-aminoacid proapelin. Immunoassays are the most widely used method to quantify apelin peptides in biological fluids. They commonly involve antibodies targeted against their common C-terminal part. Using such tests that cannot discriminate one form from the others, a wide range of plasmatic concentrations has been reported, i.e. from 20 to 4000 pg/mL. Such concentration range as well as the inability to individually quantify each form prevents the clinician from easily distinguishing healthy subjects from patients with a known heart failure and identifying a clear biomarker. Therefore, we have recently developed a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS, MRM mode) method to simultaneously quantify the 5 putative apelin peptides in human plasma. Ten different plasma samples were analyzed both with a commercial immunoassay and our mass spectrometric method. Immunoassay have revealed immunoreactive apelin concentrations around 200-400 pg/mL in theses samples. However, despite the high level of sensitivity of our approach in the pg/mL range, we were not able to detect any of the expected apelin peptide. These results question the nature and/or the concentration of circulating apelin peptides as well as the specificity of the immunoassays that have hitherto been used for clinical applications. Determination of these forms in human plasma is a particularly challenging task and is currently ongoing in our laboratory, due to the low concentrations of these peptides. An untargeted approach involving both high-resolution/high-mass accuracy mass spectrometry (LTQ-Orbitrap) and carefully optimized extraction methods has been used to identify potential circulating forms. In parallel, the same untargeted approach has been applied to bovine colostrum, a matrix known to contain high apelin levels (~ng/mL). The untargeted method efficiency was proved by highlighting 48 different endogenous apelin peptides in bovine colostrum. Eleven of those peptides were consistently observed in all the colostrums tested but each of the 48 forms has highly variable relative abundances. Based on these results and complementary data obtained following degradation of apelin-55 in plasma, we are now developing a multiplexed LC-MRM assay targeting more than 20 putative forms for a more sensitive screening of apelin peptides in human plasma. During this presentation, we will show how mass spectrometry techniques can represent useful and efficient tools to identify and further quantify biomarkers in biofluids.
301/381
[P215] Identification of liver immuno-markers in hepatocellular carcinoma

M. Ramzan1, N. Sturm1, K. Arafah2, P. Arvers3, E. Jouvin-Marche1, J-P. Zarski1, V. Leroy1, F. Berger1, P. Bullet2, P. Marche1
1
Immunologies Analytiques des pathologies chroniques, Inserm/UJF U823, Rond-point de la Chantourne, 38706 La Tronche
2 3 4 5
Plateforme BioPark, Btiment LE FORUM, 74160 Archamps Institut de recherche biomdicale des armes, CRSSA, Avenue des Maquis du Grsivaudan, 38702 La Tronche Laboratoire de Nanomdecine et cerveau, Inserm/UJF U836, Chemin Fortun Ferrini, 38706 La Tronche
AGe Imagerie et Modlisation, AGIM/UJF CNRS FRE 3405, Pavillon Taillefer, Facult de mdecine de grenoble, 38706 La Tronche Contact: K. Arafah (karim_arafah@yahoo.fr)
Keywords: Clinical proteomics, Imaging mass spectrometry Background: Hepatocellular carcinoma (HCC) incidence is increasing worldwide. Most HCC occur in chronic hepatitis associated to liver inflammation with increases of liver infiltrating lymphocytes (LILs) outside the tumor: A molecular characterization of LIL associated to HCC cirrhotic liver requires screening precise histological regions (lymphoid aggregates, portal tracts and septa). Methods: HCC livers, 18 patients infected by hepatite C virus (HCC-HCV) and 16 abusing alcohol (HCC-OH), were analyzed with anti-CD3, -CD4, -CD8 and -CD20 antibodies for determining LILs in fibrous septa (FS) and parenchymatous (Pa) nodules of cirrhotic zones (CZ). The proteomes of CZ were analyzed by SELDI-MS. Statistical tool (PLS-DA) highlighted the differential ion markers obtained in the 2 patient groups. The relevant peaks were detected by liquid chromatography (LC) MALDI-MS in tissue lysates and investigated by MALDI imaging (MSI). Results: Most of LILs found inside the FS and less abundant in the Pa. LILs were higher in the CZ of HCC-HCV than of HCC-OH group. SELDI-TOF-MS analysis showed homology of protein profiles between sample duplicates. Among 105 identified peaks, 16 were more and 9 were less abundant in HCC-HCV vs HCC-OH group. Variable Importance for the Projection (VIP) plot combining molecular and cellular parameters showed highly significant parameters including the 4 types of LILs described beyond and 2 clinical parameters. Highest VIP scores showed 3 protein ions significantly more expressed in HCC-HCV than in HCC-OH group clustered with CD3 and CD8 cells. High numbers of CD8 cells were also associated with tumor recurrence. In parallel, LC MALDI-MS and MSI on liver slices yielded to convergent data. Conclusion: Orthogonal proteomics allowed molecular identification of potential markers in HCC. MSI gives complementary results about tissue distribution of markers inside the LILs and is indicative of HCC recurrence after liver resection, providing new insights in prognosis.
302/381
[P216] Non linear effects on Kinetic Method Measurements

Sandrine Bourgoin-Voillard1, Denis Lesage1, Carlos Afonso1, Franoise Fournier1, Emilie-Laure Zins2, Claude Pepe2, Peter B. Armentrout 3, Jean-Claude Tabet1
1
Institut Parisien de Chimie Molculaire (UMR CNRS 7201) - FR 2769 UPMC univ Paris 06, 4 place Jussieu, 75005 Paris
2
Laboratoire de Dynamique Interactions et Ractivit LADIR (CNRS UMR 7075), UPMC univ Paris 06, 4 place Jussieu, 75005 Paris
3
Department of Chemistry, University of Utah, , Utah 84112 Salt Lake City, USA
Contact: Denis Lesage (denis.lesage@upmc.fr)
Keywords: Organic and inorganic mass spectrometry The original Cooks kinetic method was the landmark for comparisons of thermochemical data by tandem mass spectrometry.[1] It constitutes an elegant approach for the determination of thermochemical data such as proton affinity (PA) and gas-phase acidity (Hacid). The method is based on the dissociation of heterodimers and the PA or Hacid of functionalized compounds (A0) is obtained relative to reference compounds (Ai) with known PA or Hacid values. In the literature, several non-linear effects have been evidenced upon the use of the kinetic method. In fact it has been shown by Vkey and Drahos that the first kinetic method plot (ln(k1/k2 vs H)) is not strictly linear.[2] Such non-linearity necessarily induces a reduction in the accuracy of the measurements as they are based on linear regression analyses. This behavior appeared to be very strong for small molecules presenting a large entropy effect. This non-linear deviation is reinforced at high activation energies. Non-linear deviations have been also evidenced in the second kinetic method plot (e.g. GAapp(A0) vs Teff). These non linear effects have been interpreted as resulting from artifacts occurring at high or low energy conditions[3] or resulting from the existence of various isomeric forms within the dimer.[4] In this work, we are interested in non-linear effects occurring in the second plot used for the extended kinetic method treatment. The example discussed herein concerns Hacid determination of substituted phenols. Phenolic compounds have been investigated to determine their thermochemical properties. In particular, we report here, Hacid and S values of halogenated phenols determined by proton exchanges with carboxylic acids. In addition, experimental data have been compared with those obtained from DFT calculations and RRKM simulations using Crunch[5] and Masskinetics[6] softwares. It is shown that the approximations done and experimental conditions required to apply the extended kinetic method are not always consistent with those advised by the method. [1] Cooks RG and Kruger TL, J. Am. Chem. Soc., 1977, 99: 1279 [2] Drahos L and Vekey K, J. Mass. Spectrom., 2003; 38: 1025 Drahos L, Peltz C and Vkey K, J. Mass. Spectrom., 2004; 39: 1016 [3] Wenthold PG, J. Am. Soc. Mass Spectrom. 2000; 11, 601 [4] Fournier F, Afonso C, Fagin AE, Gronert S and Tabet JC, J Am Soc Mass Spectrom 2008; 19 (12), 1887 [5] Rodgers MT and Armentrout PB, J. Chem. Phys., 1997; 106, 4499 [6] Drahos L and Vkey K, J. Mass Spectrom., 2001; 36: 237
303/381
[P217] The Dictyostelium macropinocytic proteome: a step towards a deep analysis of macropinocytosis dynamics
Agns Journet1, Sabine Brugire1, Agns Chapel1, Syvie Kieffer1, Christophe Bruley1, Yves Vandenbrouck1, Christophe Masselon1, Grard Klein1, Laurence Aubry1
1 2 3
CEA, IRTSV, Laboratoire Biologie Grande Echelle , 17 rues des martyrs, 38054 Grenoble, France INSERM, U1038, , 38054 Grenoble, France Universit Joseph Fourier, , 38000 Grenoble, France
Contact: Sabine Brugire (sabine.brugire@cea.fr)
Keywords: Systems biology, High mass and high resolution mass Spectrometry, Bioinformatics et biostatistics dedicated to proteomics Dictyostelium discoideum is a simple haploid eukaryote that shares many functions and molecular features with macrophages and dendritic cells, in particular their endocytic capacities (phagocytosis and macropinocytosis). Endocytosis serves various functions ranging from the internalization of nutrients to the regulation of signaling cascades by altering the accessibility of membrane signaling receptors to their downstream effectors. Because of its phylogenetic proximity with higher eukaryotes and the conservation of the protein networks underlying the endocytic process, identification of the repertoire of Dictyostelium proteins residing on or associated with the macropinocytic pathway is of immediate interest to further dissect this essential mechanism. As a preliminary step to this goal, we established the inventory of proteins implicated in the macropinocytic pathway by using a magnetic purification procedure resulting in a high degree of purity, coupled to a mass spectrometry-based proteomics. Dictyostelium macropinocytic compartments were isolated after loading with colloidal iron and then purified on a magnet as described in [1]. Proteins were separated by electrophoresis on denaturing gels and subjected to in-gel trypsin digestion. MS-MS data were acquired on an LTQ-Orbitrap mass spectrometer coupled to a nano-HPLC. 103 analyses were performed including technical replicates. Mascot search results were filtered using a two-step process leading to an overall false discovery rate of 1.95% at the peptide level. Filtering and grouping of the multiple occurrences of a particular peptide led to an inventory of 18,395 distinct peptides corresponding to a final list of 2,251 non-redundant proteins, the DdEndo list (deposited in the Pride database). Bioinformatics analyses indicated that the most abundant proteins present in this list associated with signaling, vesicular trafficking and cytoskeleton. 23% featured membrane-spanning domains while around 30% were without any references, neither annotations nor associated prediction. Compared with recent studies [2, 3], the DdEndo list displays an enhanced depth of coverage, in particular for membrane-embedded proteins, validating our purification method. The building of this library of peptide identifications enables further high throughput label-free quantitative analyses and paves the way for a future organellar proteomics approach of the dynamics of macropinocytosis. 1. Aubry, L., Klein, G., Purification techniques of subcellular compartments for analytical and preparative purposes. Meth. Mol. Biol. 2006, 346, 171-185. 2. Boulais, J. et al., Molecular characterization of the evolution of phagosomes. Mol. Syst. Biol. 2010, 6, 423. 3. Gotthardt, D. et al., Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake. Mol. Cell. Proteomics 2006, 5, 2228-2243.
304/381
[P218] Chloroplast quantification: use of a software environment hEIDI - for the management and the exploration of MS/MS data
Claire ADAM1, Myriam FERRO1, Vronique DUPIERRIS1, Anouk EMADALI1, Magali COURT1, Christophe BRULEY1, Norbert ROLLAND2, Daniel SALVI2
1
CEA, iRTSV, Laboratoire dEtude de la Dynamique des Protomes, Grenoble, F-38054, France ; INSERM, U880, Grenoble, F-38054, France ; Universit Joseph Fourier, Grenoble, F-38054, France, 17 rue des Martyrs, 38000 Grenoble, France
2
CEA, iRTSV, Laboratoire de Physiologie Cellulaire Vgtale, Grenoble, F-38054, France ; CNRS, UMR5168; INRA, UMR1200, F-38000 Grenoble, France; Universit Joseph Fourier, Grenoble, F-38054, France, 17 rue des Martyrs, 38000 Grenoble, France Contact: Claire ADAM (claire.adam@cea.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics, Proteins, peptides and small molecules quantification Many proteomic investigations imply sample fractionation and/or different sample preparation, in order to get the best proteome coverage. Consequently many analyses are likely to be generated in the context of a specific project and the many corresponding results (identifications, quantification etc.) have to be compiled. In order to manage both identifications and spectral counts related to several LC-MS/MS analyses, we developed a software suite dedicated to Mascot-generated results. A first module called IRMa (Identification of Results from Mascot ; Dupierris et al ., Bioinformatics, 2009) allows both automatic and manual validation of Mascot identification results corresponding to a single analysis. Validated results can be exported either in documents (Excel or PDF formats) or in a relational database. The so-generated documents can be customized in order to contain information (list of peptides, false positive rate, annoted spectra etc.) that is compliant to proteomics journals guidelines. If IRMa validated results are stored in a relational database a second module called hEIDI (Exploration and Interpretation Data Identification) is able to handle multiple analyses. Using a user-friendly interface hEIDI allows different analyses to be compiled in order to retrieve the list of non-redundant protein groups. Moreover hEIDI allows direct comparison of groups of analyses to be performed, on the basis of the protein groups and of spectral counting. This is of particular interest if one wants to compare two pools of samples (mutant vs. wild type strains ; test of analytical conditions etc.). The versatility of such a tool will be presented through the quantitative analyses of chloroplast sub-fractions. This experiment represents around 500 LC-MS/MS analyses performed on a high resolution mass spectrometer that have been used to set up the AT_CHLORO database (http://www.grenoble.prabi.fr/at_chloro/). This database stores information for proteins identified in different sub-fractions obtained from A. thaliana chloroplasts (Ferro et al., 2010), in particular subplastidial localization. To obtain this localization, highly purified fractions of envelope, stroma and thylakoids were obtained and spectral counting was used to classify proteins according to their sub-plastidial localization. Using statistical analysis, the localization of 819 proteins, that are present in the AT_CHLORO, was determined. Link: http://biodev.extra.cea.fr/docs/heidi
305/381
[P219] High-throughput proteogenomics of an extra large group of marine microorganisms

Joseph Alexander Christie-Oleza1, Guylaine Chaussinand1, Jean Armengaud1
1
Laboratoire de Biochimie des Systmes Perturbs, CEA Marcoule, DSV, iBEB, SBTN, LBSP, BP17171, 30207 BAGNOLS-SUR-CEZE, France Contact: Joseph Alexander Christie-Oleza (joseph_christie@yahoo.es)
Keywords: Systems biology, Bioinformatics et biostatistics dedicated to proteomics The structural and functional annotation of microbial genomes is nowadays relying on automatic pipeline systems. The key for an accurate annotation consists in blending biochemical evidences to the genome interpretation. In this work we applied high-throughput proteogenomics to a member of the abundant group of marine bacteria, Roseobacters, as a seed to better annotate the whole clade. We extensively characterized the proteome of Ruegeria pomeroyi by searching over 1.1 million MS/MS spectra against a six-frame translated genome database. A large dataset of peptides (over 600,000 assigned MS/MS spectra) was obtained. We identified 2006 polypeptides among which 34 are encoded by Open Reading Frames (ORFs) that had not previously been annotated. From the pool of one-hit-wonders, i.e. those ORFs specified by only one peptide detected by tandem mass spectrometry, we could confirm the probable existence of five additional new genes after proving that the corresponding RNAs were transcribed. We also evidenced the most N-terminal peptide of 486 polypeptides from which 64 were at first badly annotated. By extending these re-annotations to the other 36 Roseobacter isolates sequenced till date (ortho-proteogenomics), we propose the correction of the assigned start codons of 1,082 homologous genes in the clade. Moreover, we evaluated the genome expression of Ruegeria pomeroyi under several natural environmental conditions revealing the versatility of the bacterium to adapt to anthropogenic influence, poor nutrient concentrations or the presence of the natural microbial community. Comparative proteogenomics consisting in the analysis of several Roseobacter strains opens new perspectives for improvements of genome annotation. Our proteogenomic efforts will increase reliability of future annotation of marine bacteria genomes.
306/381
[P220] Multiplex ligand fishing in human plasma using SUPRA-MS approach : Surface Plasmon Resonance Array Mass Spectrometry
Fabien Remy-Martin1, Marven El Osta2, Graldine Lucchi2, Rabah Zeggari1, Alain Rouleau1, Thrse Leblois1, Wilfrid Boireau1, Patrick Ducoroy2
1 2
FEMTO-ST MN2S Universit de Franche-Comt, 32 rue de l'Observatoire, 25000 Besanon CLIPP IFR100 CHU Universit de Bourgogne, 1 rue du Professeur Marion, 21000 Dijon
Contact: Marven El Osta (marven.elosta@clipproteomic.fr)
Keywords: Imaging mass spectrometry, Clinical proteomics Background: The combination of immunoassay with MS has been revisited recently and opens a promising way for the complete characterization of targeted proteins in comparison with conventional methods like ELISA (1,2,3). We recently implemented a new strategy to detect and identify proteins of interests, in multiplex format, in plasma at low femtomole level combining Surface Plasmon Resonance Array-Mass Spectrometry (MALDI) directly onto the chip without elution step. Methods: Biochip engineering has been developed to assess high level of antibody grafting in macro array format (4 x 4 spots) and prevent non-specific adsorption in complex media. The multiplexed capture analyses of antigens in plasma were performed through SPRi-Plex apparatus (HORIBA scientific). The steps of reduction, digestion of proteins captured on chip and the deposition of matrix were performed by the IMAG PREP technology. The spots of proteins and peptides were analyzed by MALDI UltrafleXtreme using the MS and MS/MS mode. Results: After optimization of procedures, the level of detection of Lag 3 antigen in plasma is 10 nM by SPRi. The robust methods of treatment of biochip after SPR analysis allow the identification and characterization of antigens by MS and MS/MS analysis. The results show a good correlation between the quantities of protein captured on the chip (~3 to 7 femtomol/mm2) and the number of peptides detected and the identification Mascot score. Conclusion: We demonstrate the capacity of this new Platform analysis Supra-MS to detect and characterize proteins in human plasma, in multiplex format. One application of this platform is the detection and quantification of human protein variants described as potential biomarker of disease. References: 1) Bellon et al, Anal Chem, 2009, 81, 7695-7702 2) Boireau et al. Biosen Bioelec, 2009, 24, 1121-1127 3) Trenchevska et al J Proteome Res, 2010, 9(11):5969-73. Link: www.clipproteomic.fr
307/381
[P221] Phosphorylation events in response of iron stress in Arabidopsis

Mathilde Decourcelle1, Valrie Rofidal1, Michel Rossignol1, Sonia Hem1, Claude Nespoulous1
1
Laboratoire de Protomique Fonctionnelle, 2 place Pierre Viala, 34060 Montpellier cedex, France
Contact: Mathilde Decourcelle (decource@supagor.inra.fr)
Keywords: Signaling, interactomics and post-translational modifications Environmental stimuli activate a wide range of signaling pathways involving reversible posttranslational modifications of proteins, leading to the cellular responses. In plants, iron is involved in most of the cellular redox reactions and in electron transfer chains. In order to both sustain growth and avoid the toxicity of free cellular iron, a fine-tuning of import, storage and remobilization to and from the ferritins, the ubiquitous iron storage proteins coded by the AtFer1 gene, is required [1]. Thus identification of the proteins playing a role in these essential processes for optimal plant productivity is of a primordial interest. An excess of iron in the medium is known to induce successive post-translational events [2], leading to the increase in the AtFer1 transcript level. Among these, a phosphorylation event was proven to occur early in the transduction sequence. Phosphorylation is one of post-translational modifications (PTM) the most involved in cell signaling and phosphoproteomics technologies recently displayed a maturity convenient for functional investigations. The progress in enrichment strategies and the powerful of mass spectrometry today allow in-depth and large-scale proteomic analysis that generate protein data sets of still more big size [3, 4]. In plant, although being yet not so large as in mammals, the reported phosphoproteomes begin to have a sufficient size [5,6,7] to undertake comparative quantitative studies in Arabidopsis allowing the identification of discrete proteins potentially involved in response to stresses. Here, we propose a workflow for both the characterization of the phosphoproteome from an Arabidopsis whole cell lysate and the analysis of its responses to a stimulus using a label-free LCMS strategy. Phosphopeptides enrichment was performed using combination of SCX and TiO2 chromatographies. The mass spectrometry analysis was done using a high resolution Q-TOF mass spectrometer coupled to a nanoflow HPLC ChipCube. With this strategy, over than 1000 phosphorylation sites were routinely identified with a high confidence score. One third of them was novel in comparison to the largest reported phosphoproteomes [5, 6]. In addition, label free quantification indicated that several sites were differentially up or down regulated after iron treatment. Results are discussed in terms of the possible involvement of identified candidates in response to this stress. 1. Briat, J.F., et al., Ann Bot (Lond), 2009. 2. Arnaud, N., et al., J Biol Chem, 2006. 281(33): p. 23579-88. 3. Lemeer, S. and A.J. Heck. Curr Opin Chem Biol, 2009. 13(4): p. 414-20. 4. Rigbolt, K.T., et al., Sci Signal, 2011. 4(164): p. rs3. 5. Sugiyama, N., et al., Mol Syst Biol, 2008. 4: p. 193. 6. Reiland, S., et al., Plant Physiol, 2009. 150(2): p. 889-903. 7. Grimsrud, P.A., et al., Plant Physiol, 2010. 152(1): p. 19-28. Link: http://www1.montpellier.inra.fr/proteome/index.htm
308/381
[P222] Insource decay et Pseudo MSn de protines par MALDI TOF iontrap
Lyna Sellami1, Claude Villard1, Therese Schembri1, Mathew E. Openshaw2, Pascale Barbier1, Omar Belgacem2, Daniel Lafitte1
1 2
plateforme protomique Timone, 27 boulevard Jean Moulin, 13005 Marseille, France Kratos Analytical - Shimadzu, Wharfside, Trafford Wharf Road, M17 1GP Manchester, UK
Contact: Lyna Sellami (lina.sellami@univmed.fr)
Keywords: High mass and high resolution mass Spectrometry, Instrumentation Lin source decay (ISD) a rcemment t applique des protines haut poids molculaire (dpassant les 60 KDa). Nous montrons une mthode peu commune qui consiste isoler des fragments issus du MALDl-ISD de lhuman serum albumin (HSA) et de lhTau40 (Tau) en induisant une fragmentation slective (jusqu' 3 cycles) tout en gardant une haute rsolution aussi bien sur lion parent slectionn que sur ses fragments. Cette technique a permis de squencer les parties N et/ou C terminales de ces deux protines. Les fragments ISD obtenus sous vide pouss (10-7 mbar) sont ensuite slectionns dans la trappe o seffectuent les cycles ms/ms suivants. Lanalyseur TOF a t employ pour acqurir les donnes en mode MS et MSn. Nous avons utilis lHSA et la Tau comme preuve de principe, la proteine totale a t soumise lISD dans le spectromtre puis les fragments ont t accumul dans la trappe avant dtre fragment. Et pour finir, lISD et la Pseudo MSn ont t ralis sur la Tau oxyde au NaOCl pour identifier une modification post traductionnelle.
309/381
[P223] Nouvelle methodologie en LC MALDI

Sega NDIAYE1, Giovanni Chiappetta1, Emmanuelle Demey1, Yann Verdier1, Iman Haddad1, Joelle Vinh1
1
Laboratoire de Spectrometrie de Masse Biologique et Proteomique, 10 rue Vauquelin , 75005 Paris
Contact: Sega NDIAYE (sega.ndiaye@espci.fr)
Keywords: Systems biology La LC MALDI n'a longtemps t que peu utilis pour l'analyse protomique en raison de sa dure trop importante, celle-ci additionnant le temps de la chromatographie avec le temps d'analyse seules quelques applications ncessitent son utilisation tel que l'iTRAQ. Nous avons valu la LC-MALDI par rapport aux techniques d'lectrospray. Nous avons montr que la combinaison d'une analyse LC MALDI avec une analyse LC-ESI permet d'augmenter la couverture de squence protique et peptidique, certains peptides tant identifis spcifiquement dans les analyses LC-MALDI. Outre ces informations complmentaires. La LC MALDI s'est rvl 2 3 fois plus rptable que l'ESI. Nous avons galement dvelopp la 2D LC MALDI et l'avons compar la 2D LC ESI avec des rsultats montrant galement une complmentarit des techniques. L'installation d'une source MALDI sur l'Orbitrap (Thermo) qui est un appareil de haute prcision et d'une grande sensibilit, a ouvert une nouvelle dimension l'analyse en LC-MALDI Cependant cette combinaison ncessite une dure d'analyse plus longue que le TOF/TOF due principalement au temps ncessaire pour raliser des MS/MS interprtables. Pour palier ce problme nous avons mis au point un moyen lgant et original d'associer les deux spectromtres de masse en combinant ce qui fait leur force, c'est dire ka prcision et la sensibilit de l'Orbitrap et la rapidit d'xcution des MS/MS pour le MALDI TOF/TOF tout ceci partir des mmes dpots.
310/381
[P224] Proteomic approaches for discovering biomarkers of diabetic nephropathy

Laurence Molina1, Randa Ben Ameur1, Nicolas Salvetat1, Capucine Bolvin1, Chamseddine Kifagi1, Fayal Jarraya2, Claude Granier1, Franck Molina1
1 2
SysDiag, UMR3145 CNRS / Bio-Rad, Cap delta, 1682 rue de la Valsire, CS 61003, 34184 Montpellier, France Nephrology Department and LIA 135, CHU Hdi Chaker, Route el Ain, 3000 Sfax, Tunisie
Contact: Franck Molina (franck.molina@sysdiag.cnrs.fr)
Keywords: Clinical proteomics, Bioinformatics et biostatistics dedicated to proteomics One of the major problems facing clinical nephrology currently throughout the world is an exponential increase in patients with end-stage renal disease (ESRD), which is largely related to a high incidence of diabetic nephropathy (e.g. 36.9% of all US patients with end-stage renal disease have a diabetic nephropathy). However, it is currently impossible to reliably predict which and when diabetic patients will develop nephropathy and progress to kidney failure. Microalbuminuria (MA), defined as urinary albumin excretion of 30-300 mg/day, is the current first clinical evidence of incipient diabetic nephropathy. But recent studies have proved it has inadequate specificity. Proteomics has evolved into a high-throughput, analytical discipline used to analyze complex biological data sets. Since many years now, many proteomic studies on the discovery of new markers of diabetic nephropathy were conducted. it is hoped that these studies will unveil biomarkers earlier and more specific than microalbuminuria is. The aim of the present work is to comprehensively review, especially in terms of biostatistics and bioinformatics analysis, the available proteomics studies devoted to DN biomarker discovery. Link: http://www.sysdiag.cnrs.fr/
311/381
[P225] Dveloppements mthodologiques en analyse protomique pour ltablissement du protome membranaire de cellules souches cancreuses issues de gliomes
Sarah Lennon1, Jean-Michel Saliou1, Emilie Audran2, Herv Chneiweiss3, Christine Carapito1, Alain Van Dorsselaer1, Jacques Haiech2, Sarah Sanglier-Cianfrani1
1
Laboratoire de Spectromtrie de Masse BioOrganique (LSMBO), IPHC, CNRS UMR7178, Universit de Strasbourg, ECPM - Bat. R5.0 - 25 rue Becquerel, 67087 Strasbourg
2 3
Institut Gilbert Laustriat, CNRS UMR 7175, Universit de Strasbourg, , 67400 Illkirch Plasticit gliale et tumeurs au cerveau, Inserm UMR 894, Universit Paris Descartes, , Paris
Contact: Sarah Sanglier-Cianfrani (sarah.cianferani@unistra.fr)
Keywords: Clinical proteomics, Bioinformatics et biostatistics dedicated to proteomics, Systems biology Les gliomes concernent lensemble des tumeurs du systme nerveux central. Malgr les avances en chirurgie mdicale et dans les thrapies mdicamenteuses, ces tumeurs restent difficiles traiter. Trs rcemment, la prsence de cellules souches cancreuses (CSC) au sein de ces tumeurs a t mise en vidence (1). Du fait de leur rsistance aux traitements de chimio/radio-thrapies actuels et de leur capacit sommeiller pendant une longue priode de temps, les CSC pourraient tre lorigine des rechutes et sont donc des cibles intressantes pour la recherche de nouveaux biomarqueurs thrapeutiques (1). En effet, jusqu prsent, les protines identifies en tant que biomarqueurs de CSC ne sont pas rellement pertinentes. Par exemple, le CD133, potentiel biomarqueur de CSC du gliome, nest exprim que dans 50 % des cas (2). Lobjectif global de ce projet est la recherche de biomarqueurs de CSC du gliome. Cette premire partie de notre tude a pour but de raliser une cartographie la plus complte possible du protome membranaire dune ligne de CSC du gliome (TG01) par protomique. Lapproche dveloppe se base sur une sparation des protines sur gel 1D, suivie par une analyse nanoLC-MS/MS. Chaque tape de cette stratgie a t optimise afin dassurer un maximum didentifications avec un niveau de confiance lev. Une attention particulire a t porte la prparation de lchantillon (analyse de la prparation membranaire avec et sans fractionnement par prcipitation au sulfate dammonium) et la rptabilit des analyses nanoLC-MS/MS. Lintrt de lutilisation de deux moteurs de recherche (Mascot et Omssa) pour augmenter la confiance accorde aux identifications en analyse protomique est galement prsente. La combinaison de toutes ces analyses a permis didentifier plus de 2900 protines dont 599 ont une annotation plasma membrane . Parmi ces dernires, 61 clusters de diffrentiation, molcules de surface intressantes pour leur application en clinique, ont t identifis. Cette tude a permis daugmenter la sensibilit de dtection par rapport des tudes protomiques prcdentes: plusieurs protines, telles que EGFR et CD221, qui navaient jamais t vues prcdemment3, mais, dont le rle important dans les gliomes a t dmontr par immunohistochimie, ont pu tre dtectes. Ces premiers rsultats de protomique seront compars aux rsultats danalyses transcriptomiques. 1.Clevers, H., Nat Med 17, 313-319 (2011). 2.Patru, C. et al., BMC Cancer 10, 66 (2010). 3.Deighton, R.F. et al. Brain Pathol 20, 691-703 (2010).
312/381
[P226] Dveloppement de micro-nano-technologies pour l'interface cerveaubiomarqueurs

Affif Zaccaria1, Ali Bouamrani1, Franois Berger1
1
GIN Inserm U836, Chemin Fortune ferrini, 38700 La Tronche
Contact: Affif Zaccaria (zaccaria.affif@gmail.com)
Keywords: Clinical proteomics Lvolution des outils danalyse protomique, en particulier le dveloppement de la spectromtrie de masse (MS), a permis de raliser des progrs majeurs dans ltude de protomes complexes. La forte sensibilit de dtection et la rapidit danalyse expliquent lengouement actuel pour cette technologie comme outil de recherche et didentification de biomarqueurs protiques pathologiques. La dcouverte de ces biomarqueurs est un enjeu biomedical crucial pour : - un diagnostic prcoce - le monitoring dune pathologie - le suivi thrapeutique ouvrant ainsi la voie une mdecine plus personnalise. La mise en vidence de ces biomarqueurs passe idalement par lanalyse tissulaire qui permet d'accder aux informations biologiques pertinentes provenant directement du foyer pathologique. Dans les pathologies crbrales, face linaccessibilit du tissu et linvasivit des biopsies, la recherche de biomarqueurs se focalise sur lanalyse des fluides biologiques priphriques, se retrouvant confronte la complexit et la grande gamme dynamique de ces fluides. Les stratgies actuelles reposant sur le pr-fractionnement de l'chantillon sont peu compatibles avec la protomique clinique haut dbit. Pour rpondre ces limitations, des ruptures technologiques permettant d'adapter ces approches aux impratifs cliniques sont indispensables. Pour cela les micro-nano-technologies apportent des solutions alternatives prometteuses. Nous avons ainsi montr que l'utilisation de nanobilles magntiques limite de manire significative la capture des protines majoritaires et apporte une solution pour la capture de sous-protomes minoritaires en fonction de leur taille et des modifications chimiques de surface. Le dveloppement de structures associant ces nanobilles et un polymre biocompatible permettant ainsi un contact entre les billes et le sous-protome circulant, constitue un vritable bioracteur dinteraction et de capture in vivo. De plus, la reproductibilit observe lors d'une analyse haut dbit en font un outil adapt la protomique clinique. En parallle, le dveloppement et l'utilisation d'un outil d'empreinte tissulaire bas sur la technologie du silicium a permis de mettre en vidence la capture de protines crbrales en limitant considrablement la lsion tissulaire occasionne par rapport une biopsie conventionnelle. L'ingnierie de l'outil adapt aux outils de micro-chirurgie actuels permet de raliser des empreintes tout en conservant la spatialit des structures crbrales abordes lors des interventions chirurgicales sans modifier les procdures standards.
313/381
[P227] Using mass spectrometry to find partners of the thyroid sodium/iodide symporter (NIS).
Didier MARCELLIN1, Jean-Charles GAILLARD2, Elisabeth DARROUZET1, Jean ARMENGAUD2, Thierry POURCHER3
1
Laboratoire des Transporteurs en Imagerie et Radiothrapie en Oncologie, SBTN, iBEB, Centre Atomique de Marcoule, 30207 Bagnols sur Cze
2
Laboratoire de Biochimie des Systmes Perturbs, SBTN, iBEB, Centre Atomique de Marcoule, 30207 Bagnols sur Cze
3
Laboratoire des Transporteurs en Imagerie et Radiothrapie en Oncologie, SBTN, iBEB, UNSA, CAL, Facult de mdecine, 28 av de Valombrose, 06107 Nice Contact: Didier MARCELLIN (didier.marcellin@cea.fr)
Keywords: Systems biology, Signaling, interactomics and post-translational modifications NIS is a membrane protein responsible for the accumulation of iodide into the thyroid follicle. Iodide is then used for T3 and T4 hormones synthesis. NIS properties are also widely used for the radiotherapy of thyroid cancers and their metastases, and its expression by gene therapy could widen even more its medical application. In our laboratory we are interested in studying human NIS post-translational regulation. Indeed, the functional expression of this symporter is finely regulated by a control of its subcellular localization and its recycling. Such regulation most likely involves interactions with other proteins. In order to identify such potential partners we decided to use crosslink on whole cells overexpressing hNIS in order to freeze the interactions. This step is followed by immunoprecipitation using in-house monoclonal antibodies against hNIS, and subsequent analysis by mass spectrometry. Different aspects of this protocol will be discussed including comparative studies of various magnetic beads for immunoprecipitation, and preliminary results will be presented. The next step will consist in applying this modus operandi directly on human thyroid tissue samples, cancerous or not, from the Antoine Lacassagne Center tissues library in Nice (France).
314/381
[P228] Use of False Detection Rate in proteomic data merging and optimization of the selection of identification results
Franois Allain1, Jean Armengaud1, Olivier Pible1
1
CEA, DSV, IBEB, SBTN, Laboratoire de Biochimie des Systmes Perturbs, CEA Valrho, BP17171, 30207 Bagnols sur Ceze Contact: Olivier Pible (olivier.pible@cea.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics Identification results based on MS/MS spectra assignments for large scale proteomic experiments must be provided with a measure of identification certainty such as a False Detection Rate (FDR) as specified in the current guidelines of the Mol Cell Proteomics journal. The estimation of False-positives in a large experiment is not trivial. We designed a tool, called FDRfox, which will allow the merging of different datasets at a given confidence level. We rely here on results provided by the Mascot engine (Matrix Science) using a decoy database. Setting as initial input a final protein FDR value, the tool will use a uniform peptidic FDR level between datasets, rather than a uniform p_value (significance threshold in the Mascot software). This will immunize from inter-assay fluctuations. Protein FDR will either be based on proteins identified with 1 or 2 peptides. FDRfox is seamlessly interfaced with Mascot, and will provide the individual p_values of each data subset resulting after merging in the global protein identification results at the target protein FDR.
315/381
[P229] N-terminal oriented proteogenomics of Roseobacter desnitrificans

Cline Bland1, Joseph A. Christie-Oleza1, Emie Durighello1, Bernard Fernandez1, Jean-Charles Gaillard1, Jean Armengaud1
1
Laboratoire de Biochimie des Systmes Perturbs, SBTN, iBEB, DSV, CEA Marcoule BP17171, 30207 Bagnols sur Cze cedex Contact: Cline Bland (celine.bland@cea.fr)
Keywords: Proteins, peptides and small molecules quantification High-throughput proteomic data can be used to verify the coding regions of a genomic sequence and refine its annotation (Armengaud, 2009). One of the main problems in automatic annotation is the identification of the start codons, being the estimated error rate superior to 10%. Thus, start codon reannotation becomes one of the main issues of proteogenomics. Labelling the N-terminus of intact proteins using N-tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu) has proved a reliable method to ascertain protein N-termini (Gallien et al., 2009). The advantages of TMPP-derivatized Nterminal peptides are i) an increase of the hydrophobic character of the product, increasing its retention time in reverse phase chromatography, ii) an improvement of their ionization efficiency due to the introduction of a fix positively charged group, and iii) a much simplified fragmentation pattern that must be considered during analysis. After a first application on the Deinococcus deserti bacterium where non-canonical translation initiation codons have been discovered (Baudet et al., 2010), we analyzed another bacterium, namely Roseobacter desnitrificans, a specie belonging to the Roseobacter clade, which is a key component of surface marine ecosystems. Our first results show that amongst 291 unique proteins with N-terminal signature obtained by MS/MS, 212 were observed with a TMPP label. Specific exploration of non-canonical translation initiation codons will be performed with larger datasets.
316/381
[P230] VUV-photodissociation of peptides in the gas phase probed by synchrotron radiation

Francis Canon1, Aleksandar Milosavljevic1, Pascale Sarni-Manchado4, Vronique Cheynier 4, Guillaume Van der Rest5, Laurent Nahon1, Alexandre Giuliani1
1 2 3 4 5
Synchrotron SOLEIL, Lorme des Merisiers, 91192 Gif-sur-Yvette INRA, U1008, CEPIA, , 44316 Nantes Institute of Physics Belgrade, University of Belgrade, Pregrevica118, 11080 Belgrade INRA,UMR1083 SPO, 2, place Viala, 34060 Montpellier Laboratoire DCMR, UMR 91128, Ecole Polytechnique, 91128 Palaiseau
Contact: Francis Canon (francis.canon@synchrotron-soleil.fr)
Keywords: Organic and inorganic mass spectrometry, Instrumentation Determination of peptide sequence is of paramount importance in proteomics and in studies regarding proteins. The quest for an efficient technique of fragmentation that is not influenced by the peptidic sequence is still relevant today. To address this issue, we have developed a new technique of tandem mass spectrometry. We present here our first results regarding this new technique called dissociative photoinization (DPI). Briefly, a Thermo LTQ XL ion trap was coupled to a VUV beamline at the SOLEIL synchrotron radiation facility. Monochromatized photons were introduced into the ion trap, thus allowing the ion of interest to be irradiated. We have studied the fragmentation of the Substance P induced by this technique. Substance P is a 11 amino-acid peptide (1347Da), which has been often used by the past as a model peptide. As a consequence, its pattern of fragmentation induced by different technique of tandem mass spectrometry is well-documented 1,2. Our investigation has been pursued on a model protein, IB5, which is a human salivary proline rich-protein whose function is to scavenge tannins 3. IB5 is a 70 amino-acid protein with a molecular weight of 6923Da.5 To study the ability of this technique to localize posttranslational modifications, we have recorded the MS/MS spectrum of different modified peptides. These results are the first report on photoionization of kDa species in the gas phase. Our data are discussed with respect to previous electron impact ionization measurements 1,4. These results demonstrate the main advantages of this technique, which are the quantity of analytical information that it provides, the ability to generate ion fragments from peptides with low charge-state and the capacity to localize posttranslational modifications (PTMs). (1) Fung, Y. M. E.; Adams, C. M.; Zubarev, R. A. J. Am. Chem. Soc. 2009, 131, 9977-9985. (2) Reilly, J. P. Mass Spectrom. Rev. 2009, 28, 425-447. (3) Canon, F.; Giuliani, A.; Pat, F.; Sarni-Manchado, P. Analytical and Bioanalytical Chemistry 2010, 398, 815-822. (4) Budnik, B. A.; Tsybin, Y. O.; Hkansson, P.; Zubarev, R. A. J. Mass Spectrom. 2002, 37, 11411144. (5) Canon, F.; Pat, F.; Meudec, E.; Marlin, T.; Cheynier, V.; Giuliani, A.; Sarni-Manchado, P. Analytical and Bioanalytical Chemistry 2009, 395, 2535-2545.
317/381
[P231] Liquid Extraction Surface Analysis (LESA) combined with nESI-MS for direct sampling of planar tissues
Reinaldo Almeida1, Daniel Eikel3, Simon Prosser3, Jack Henion3, Christer Ejsing1
1
University of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense, Denmark
2 3
Advion Biosciences Ltd, Koa Hockham Building, CM20 2NQ Harlow,UK Advion BioSystems, Inc, 19 Brown Road, 14850 Ithaca NY , USA
Contact: Reinaldo Almeida (almeidar@advion.com) Keywords: Imaging mass spectrometry, Instrumentation, Separative analysis methods The direct analyses of biological tissues require atmospheric pressure surface sampling/ionization of small sample volumes, where analytes can be directly analyzed from a variety of surface types without sample preparation. Liquid extraction surface analysis combined with nano-electrospray mass-spectrometry (LESAnESI-MS) is a complementary analytical approach to other surface-oriented MS methods such as DESI, LDI, DART or MALDI imaging MS, where a liquid micro junction surface sampling probe is used to extract the analyte directly from a surface. A small solvent droplet is placed on the tissue area of interest for analyte extraction and then aspirated into a conductive pipette tip for automated chip based nano electrospray infusion using the Advion TriVersa Nanomate. Here we applied LESA to different surfaces like whole body animal slices in early stage toxicology studies, Dried Blood Spot Cards (DBS) for drug and drug metabolite monitoring studies and Thin layer chromatography (TLC) for determination of small drug molecules. Thin mouse tissue slices pre dosed with sulforafane or propranolol, were investigated for the analysis of drug and drug metabolite distribution of whole body and compared with control tissue. The parent drug ( SFN / Propranolol) and the phase II metabolites (SFN-GSH,SFN-NAC / Hydroxypropranolol-glucuronide) were screened in MRM mode in different thin tissue sections ( lung, liver, kidney, brain ) from the dose and control mouse. Excellent signal to noise ratio could be achieved. For the analyses of drug and drug metabolites from Dried Blood Spot Cards, standards of small drug molecules were spiked to blood samples and spotted in 15ul aliquots onto Whatman DBS collection cards and allowed to dry. Using a 10 compound mix as well as these compounds individually we were able to demonstrate a LOQ of 10-100 ng/mL depending upon the nanoelectrospray response of the selected compounds. The optimized extraction-spray solvent was determined to be 80% Methanol: 20% Water + 0.1% Formic Acid. This solvent mixture provided a satisfactory compromise between efficient surface extraction of the drugs from the DBS matrix as well as providing good spray characteristics for the nanoelectrospray experiments. For the determination of Small Drug Molecules from Thin Layer Chromatography Plates Venlafaxine and its Odesmethylvenlafaxine and N-desmethylvenlafaxine metabolites were selected as test articles. 500 ng levels of these three compounds were applied at the origin of a silica gel TLC plate (Merck). From these experiments it was demonstrated that the applied 500 ng levels of each of these small drug molecule compounds readily produced acceptable MS and MS/MS mass spectra data allowing confirmation of their known structures. These same three compounds were added to control human urine and cleaned up by C-18 SPE to provide a series of urine extracts. The observed spots on the TLC Plate for each compound were then robotically 'sampled' by the NanoMate and their corresponding nano electrospray SRM transitions (m/z 279>260>215; m/z264>246>201, and m/z 265>246>215, respectively, were monitored by MS/MS/MS using a Bruker HCT ion trap. Under these conditions acceptable MS/MS/MS data to 100 ng/mL concentration were achieved. Link: www.advion.com
318/381
[P232] Analyse protomique des complexes HIF2 dans les mlanomes : identification de rgulateurs cls du dveloppement mlanocytaire.
Anne-Lise Steunou1, Manuelle Ducoux-Petit 3, Eric Clottes2, Anne Gonzalez-de-Peredo3, Bernard Monsarrat3, Monique Erard2, Odile Burlet-Schiltz3, Laurence Nieto4
1
Laboratoire Cancer, Centre de Recherche du Centre Hospitalier Universitaire de Qubec, 9, rue Mc Mahon, 2784-2, 2784-2 Qubec, Canada, G1R 2J6
2
Biophysique Structurale, Institut de Pharmacologie et de Biologie Structurale, UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex 4
3
Protomique et Spectromtrie de Masse des Biomolcules, Institut de Pharmacologie et de Biologie Structurale, UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex 4
4 5
Universit Toulouse III - Universit Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9
Microenvironnement, Cancer et Adipocyte, Institut de Pharmacologie et de Biologie Structurale, UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex 4 Contact: Manuelle Ducoux-Petit (manuelle.ducoux@ipbs.fr) Keywords: Signaling, interactomics and post-translational modifications, Proteins, peptides and small molecules quantification , Bioinformatics et biostatistics dedicated to proteomics De faibles pressions en oxygne sont une caractristique commune aux tumeurs solides. Lhypoxie locale contribue la progression tumorale et diminue la rponse aux radio et chimiothrapies. Les facteurs de transcription HIF (Hypoxia Inducible Factor) sont des rgulateurs cls de ladaptation cellulaire lhypoxie dans les cellules normales mais galement dans des conditions pathologiques telles que le dveloppement tumoral. Les protines HIF sont htrodimriques : une sous-unit alpha(type 1 ou type 2) rgule par la pression partielle en oxygne, associe HIF-1beta, exprime de faon constitutive. Les protines HIF sont impliques dans la transformation oncognique des mlanomes (1), et certaines tudes ont montr que leur surexpression dans les mlanomes cutans tait corrle un mauvais pronostic vital pour les patients (2). Dans le but de mieux comprendre limplication des protines HIF dans le dveloppement des mlanomes, nous avons cherch mettre en vidence les partenaires spcifiques des protines HIF (1 et 2) dans ce contexte cellulaire. Pour cela, des lignes de mlanome (501-Mel) surexprimant de faon stable soit HIF-2alpha (ou HIF-1alpha) soit le vecteur vide ont t tablies. La stratgie utilise combine une immunoprcipitation des complexes contenant HIF-2alpha suivie d'une analyse protomique quantitative diffrentielle label-free (contre une immunoprcipitation contrle) sur un LTQ-Orbitrap XL (3). Des partenaires connus des HIF ont t ainsi isols (HIF-1beta ou le co-activateur transcriptionnel P300) mais ce travail a surtout permis de mettre en vidence de nouveaux interactants parmi lesquels un grand nombre de rgulateurs de la transcription. Parmi eux, valids par des expriences de co-immunoprcipitations, se trouvent des acteurs majeurs de la rgulation de l'expression gnique dans les mlanomes : MITF (Microphthalmia-associated Transcription Factor) et Sox10, rgulateurs cl de la mlanogense, la protine beta-catnine, protine drgule dans environ 30% des mlanomes ainsi que TFAP2A, qui a un rle lors de la transformation de mlanocyte en mlanome. Ces protines sont impliques dans le contrle de la prolifration ou de la diffrenciation cellulaire. Linteraction entre HIF-2alpha et ces protines saccompagne dun phnotype hyper-pigment et dun retard de prolifration cellulaire. Ltude de linteractome de HIF-1alpha a mis en vidence que deux des partenaires identifis pour HIF2alpha, Sox10 et TFAP2A ninteragissent pas avec HIF-1alpha. De plus, les modifications de pigmentation et de croissance cellulaire observes chez les cellules surexprimant HIF-2alpha, ne sont pas prsentes dans les cellules surexprimant HIF-1alpha. Ces rsultats suggreraient un effet anti-tumoral pour HIF-2alpha et protumoral pour HIF-1alpha. Linjection en sous-cutane de cellules surexprimant soit HIF-1alpha soit HIF-2alpha des souris Nude pourrait alors confirmer ou infirmer notre hypothse.
319/381
[P233] Caractrisation et localisation d'insaturations de cramides par cationisation aux adduits cuivrique en ESI/MS
Danielle LIBONG1, carlos Afonso2, Denis LESAGE2, Sandra ALVES2, Arlette BAILLET1, Pierre CHAMINADE1, Jean Claude TABET2
1
Groupe de Chimie Analytique EA4041de l'Universit Paris SUD, Facult de Pharmacie, 2 rue jean Baptiste Clment, 92220 Chtenay Malabry
2
UMPC-Universit pierre et Maris Curie, 4, place de Jussieu , 75252 Paris
Contact: Danielle LIBONG (danielle.libong@u-psud.fr)
Keywords: High mass and high resolution mass Spectrometry, Organic and inorganic mass spectrometry, Instrumentation Les lipides sont des molcules essentielles la vie de la cellule, ils agissent diffrents stade : protection, compartimentation, signalisation, trafic membranaire et rserves dnergie. Pour ces raisons la lipidomique est un domaine de recherche en pleine expansion, qui offre loccasion unique danalyser le rle complexe des lipides dans le processus cellulaire. La diversit molculaire des lipides est trs importante, si lon considre la complexit des membranes biologiques et la grande varit des molcules de signalisation. Il existe diffrentes classes lipidiques qui se caractrisent par la nature de leur tte polaire tel que les triglycrides, phospholipides ou cramides. Chaque lipide prsente un grand nombre despces molculaires qui se diffrencient par la longueur de la chaine dacide gras et le nombre dinstaurations. Or il a t dmontr limportance du positionnement et du nombre dinstaurations sur lactivit biologique des lipides. Lintrt pour la localisation des doubles liaisons contenues dans les chanes dacide gras nest pas nouveau. De nombreuses mthodes de caractrisation de la localisation des doubles liaisons ont t rfrences dans la littrature, les plus classiques seffectuent en CPG-MS. Parce que la majorit des lipides complexes sont peu volatils, il est souvent ncessaire dhydrolyser la tte polaire. En MS/MS il est possible de localiser les insaturations partir dions ngatifs par collisions de haute nergie par un processus charge pige. Le but de cette tude est de dvelopper une mthode rapide didentification de lensemble des classes lipidiques en LC/ESIMS. Par le biais de la cationisation en mode lectrospray il est possible daugmenter dune part le rendement dionisation des lipides apolaires difficilement dtectables en lectrospray et dautre part de caractriser le positionnement et le nombre dinsaturations sur les chaines dacides gras. Une mthode utilisant les adduits cuivriques en ESI a t dveloppe sur diffrents cramides, lipides trs impliqus dans les phnomnes de mort cellulaire et de compartimentation du stratum cornum. Cette mthode a permis la caractrisation et le positionnement du nombre dinsaturations sur les chaines dacide gras. Lobjectif est dadapter cette mthode toute les classes de lipide afin quelle puisse tre utile un vaste champ dapplication allant de ltude de la composition membranaire des microorganismes en parasitologie jusqu diagnostic de maladies mtaboliques et gntiques
320/381
[P234] Mise au point d'une analyse quantitative label-free cible disoformes de protines dans un chantillon enrichi
Lala Sago1, Celia Dechavanne3, Florence Migot-Nabias3, Virginie Salnot1, Marjorie Leduc1, Patrick Mayeux1, Franois Guillonneau1
1 2 3 4
Plateforme Protomique Universit Paris Descartes (3P5), 22 rue Mchain, 75014 Paris INSERM u1016, 22 rue Mchain, 75014 Paris IRD, UMR 216 (IRD/UPD), 4 avenue de l'Observatoire, 75006 Paris CNRS UMR 8104, 22 rue Mchain, 75014 Paris
Contact: Franois Guillonneau (francois.guillonneau@inserm.fr)
Keywords: Proteins, peptides and small molecules quantification , Separative analysis methods, Instrumentation La quantification sans marquage par nanoLC-MS est une application rcente de la spectromtrie de masse aux projets de recherche. Le dveloppement de cette approche, partir dun modle de quantification bas sur la dtection et la quantification disoformes de protines dintrt diagnostique nous a permis de comprendre les paramtres qui entrent en jeux dans ce type dapproche. La quantification label-free repose sur la comparaison de lintensit des signaux obtenus au niveau MS pour un temps de rtention donn pour chacun des chantillons comparer. Un logiciel adapt ralise la comparaison dvnements (m/z associ un temps de rtention) au sein de diffrents chantillons en les alignant dans la dimension du temps de rtention. Pour lapproche label-free, il est ncessaire que le systme soit optimis. Nous avons donc evalu diffrents paramtres. Lanalyse de rplicats a mis en vidence la reproductibilit de notre systme notamment en ce qui concerne la chromatographie Ainsi, travers les diffrentes analyses exposes, nous avons pu montrer que le couplage nRSLC-LTQ orbitrap velos utilis au sein de la plateforme tait en adquation avec les besoins de ce type de quantification condition de dfinir une stratgie danalyse solide. Les rsultats obtenus montrent quil est possible de quantifier les diffrentes isoformes dune mme protine si tant est que les peptides discriminants sont accessibles, moyennant des optimisations denrichissement et de digestion. Link: http://3p5.medecine.univ-paris5.fr/
321/381
[P235] Liquid Extraction Surface Analysis (LESA) combined with nESI-MS for direct sampling of planar tissues
Reinaldo Almeida1, Daniel Eikel3, Simon Prosser3, Jack Henion3, Christer Ejsing1
1
University of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense, Denmark
2 3
Advion Biosciences Ltd, Koa Hockham Building, , CM20 2NQ Harlow,UK Advion BioSystems, Inc, 19 Brown road, 14850 Ithaca NY , USA
Contact: Reinaldo Almeida (almeidar@advion.com) Keywords: Systems biology, Instrumentation, Systems biology The direct analyses of biological tissues require atmospheric pressure surface sampling/ionization of small sample volumes, where analytes can be directly analyzed from a variety of surface types without sample preparation. Liquid extraction surface analysis combined with nano-electrospray mass-spectrometry (LESAnESI-MS) is a complementary analytical approach to other surface-oriented MS methods such as DESI, LDI, DART or MALDI imaging MS, where a liquid micro junction surface sampling probe is used to extract the analyte directly from a surface. A small solvent droplet is placed on the tissue area of interest for analyte extraction and then aspirated into a conductive pipette tip for automated chip based nano electrospray infusion using the Advion TriVersa Nanomate. Here we applied LESA to different surfaces like whole body animal slices in early stage toxicology studies, Dried Blood Spot Cards (DBS) for drug and drug metabolite monitoring studies and Thin layer chromatography (TLC) for determination of small drug molecules. Thin mouse tissue slices pre dosed with sulforafane or propranolol, were investigated for the analysis of drug and drug metabolite distribution of whole body and compared with control tissue. The parent drug ( SFN / Propranolol) and the phase II metabolites (SFN-GSH,SFN-NAC / Hydroxypropranolol-glucuronide) were screened in MRM mode in different thin tissue sections ( lung, liver, kidney, brain ) from the dose and control mouse. Excellent signal to noise ratio could be achieved. For the analyses of drug and drug metabolites from Dried Blood Spot Cards, standards of small drug molecules were spiked to blood samples and spotted in 15ul aliquots onto Whatman DBS collection cards and allowed to dry. Using a 10 compound mix as well as these compounds individually we were able to demonstrate a LOQ of 10-100 ng/mL depending upon the nanoelectrospray response of the selected compounds. The optimized extraction-spray solvent was determined to be 80% Methanol: 20% Water + 0.1% Formic Acid. This solvent mixture provided a satisfactory compromise between efficient surface extraction of the drugs from the DBS matrix as well as providing good spray characteristics for the nanoelectrospray experiments. For the determination of Small Drug Molecules from Thin Layer Chromatography Plates Venlafaxine and its Odesmethylvenlafaxine and N-desmethylvenlafaxine metabolites were selected as test articles. 500 ng levels of these three compounds were applied at the origin of a silica gel TLC plate (Merck). From these experiments it was demonstrated that the applied 500 ng levels of each of these small drug molecule compounds readily produced acceptable MS and MS/MS mass spectra data allowing confirmation of their known structures. These same three compounds were added to control human urine and cleaned up by C-18 SPE to provide a series of urine extracts. The observed spots on the TLC Plate for each compound were then robotically 'sampled' by the NanoMate and their corresponding nano electrospray SRM transitions (m/z 279>260>215; m/z264>246>201, and m/z 265>246>215, respectively, were monitored by MS/MS/MS using a Bruker HCT ion trap. Under these conditions acceptable MS/MS/MS data to 100 ng/mL concentration were achieved. Link: www.advion.com
322/381
[P236] Etude protomique de deux lignes divergentes de porc (CMJR) et mise en relation avec les qualits sensorielles de la viande de porc.
Thierry Sayd1, Sylvie Blinet1, Philippe Gatellier1, Christophe Chambon2, Bndicte Lebret3, Helene Gilbert4
1 2 3 4
QuaPA-BPM, INRA de Theix, 63122 St Genes Champanelle PFEM, INRA de Theix, 63122 St Genes Champanelle SENAH, INRA St Gilles, 35590 St Gilles LGC, INRA chemin de Borde-Rouge, 31326 CASTANET-TOLOSAN CEDEX
Contact: Thierry Sayd (thierry.sayd@clermont.inra.fr)
Keywords: Systems biology Les qualits de la viande sont des caractres complexes reposant sur les proprits physiologiques du muscle et en particulier sur les protines qui le composent. Lanalyse protomique permet de mieux comprendre les mcanismes biologiques qui sous-tendent les qualits et fournit des indicateurs permettant de les prdire. Une slection divergente sur la CMJR (consommation moyenne journalire rsiduelle) chez le porc Large White, a t entreprise lINRA. Ds la gnration 3, des rponses la slection ont t observes sur les caractres de composition corporelle et de qualit technologique de la viande, la ligne CMJR- (animaux sous-consommateurs ) prsentant des pourcentages de longe et de jambon suprieurs, ainsi quun pH ultime plus bas et une luminance de la viande accrue, soit une moindre qualit technologique de la viande, relativement la ligne CMJR+. Lobjectif de notre tude est de quantifier limpact de la slection sur la CMJR sur les composantes technologiques et sensorielles de la qualit de la viande (longe) et de les relier aux proprits musculaires : composition biochimique du tissu musculaire et de la viande aprs maturation laide de lanalyse protomique du muscle Longissimus afin didentifier des voies mtaboliques explicatives des diffrences musculaires biochimiques ou sensorielles entre lignes. Dans un deuxime temps nous mettrons en relation les profils protiques obtenus des critres doxydation (protines/lipides) influenant les qualits sensorielles des viandes. Ce travail se fonde sur une population exprimentale constitue de 48 porcs appartenant aux deux lignes CMJR. Sur ces animaux, nous avons ralis plusieurs mesures (chimiques, physiques et sensorielles) pour la caractrisation des qualits de la viande. Des gels dlectrophorse 2D ont t raliss sur la fraction la plus soluble des protines des muscles longissimus dorsi de ces 48 animaux. Lanalyse des gels a t ralise laide du logiciel SamSpots. Dans un premier temps, nous avons compars les profiles protiques de ces deux lignes par ANOVA. Puis nous avons explor par analyse des corrlations, les liens entre les profils protiques obtenus avec des mesures doxydation (Taux de Carbonyles Protiques et TBA) ralis aprs conservation et chauffage sur ces 48 animaux. Les spots dintrt ont t identifis par spectromtrie de masse. Une interprtation biologique des rsultats a t propose.
323/381
[P237] Dveloppement plasmatique
dune
mthode
de
caractrisation
du
peptidome
Delphine CENTENO1, Didier VIALA1, Thierry SAYD2, Jrmy PINGUET4, Norredine HAFNAOUI 3, Claudia BICH1, Estelle PUJOS-GUILLOT 1, Christophe CHAMBON 1
1
Plateforme dExploration du Mtabolisme (PFEM), INRA de Clermont-Ferrand, site de Theix, 63122 SaintGens Champanelle
2 3 4
UR370 QuaPA, INRA de Clermont-Ferrand, site de Theix, 63122 Saint-Gens Champanelle UMR1019 NH, INRA de Clermont-Ferrand, site de Theix, 63122 Saint-Gens Champanelle Laboratoire de Pharmacologie, CHU Gabriel-Montpied, 58 Rue Montalembert, 63000 Clermont-Ferrand
Contact: Christophe CHAMBON (christophe.chambon@clermont.inra.fr)
Keywords: Separative analysis methods, Clinical proteomics Le plasma, et plus particulirement le protome plasmatique, fait lobjet dune attention particulire, avec de nombreux projets qui tentent de le caractriser et de mettre en vidence des biomolcules dintrts. Cest un dfi difficile en raison de la complexit du mlange et, ce jour, encore trs peu de travaux ont vritablement aboutis la dcouverte et la validation de biomarqueurs pouvant tre exploits grande chelle. Ltude dune sous-fraction de ce protome plasmatique est une solution pour accder un champ exploratoire moins complexe. Ainsi, nous avons choisi dtudier la fraction protomique de faible masse molculaire du plasma, appele le peptidome plasmatique. Nanmoins, apprhender cette fraction reste encore un challenge important car la fraction peptidomique est en mlange avec une quantit importante dautres molcules et en premier lieu, les protines qui sont trs majoritaires. Dans cette tude, nous avons valu si la mthode de prcipitation des protines utilise classiquement dans les problmatiques de dosages des petites molcules dans le plasma, peut galement tre utilise comme mthode de rfrence pour ltude du peptidome. Pour cela, nous lavons compare avec des mthodes simples de prparation comme lultrafiltration ou la purification SPE. Les diffrentes fractions peptidomiques obtenues sont analyses par spectromtrie MALDI-TOF (DE-PRO, ABSciex) et ESI-IT (LTQ Velos,ThermoFisher). Les rsultats obtenus sont tudis la fois sur lexhaustivit de lempreinte peptidique obtenue mais aussi dans le cadre de la mise au point dune tude encore plus spcifique sur les peptides phosphoryls du peptidome. Nous prsenterons un workflow le plus appropri permettant la fois dobtenir efficacement un sous-protome peptidique mais aussi un sous-peptidome des peptides phosphoryls avec lutilisation dune micro-colonne contenant une rsine de dioxyde de titanium. Ce phospho-peptidome apparat trs intressant car le rle des peptides phosphoryls librs dans la circulation sanguine est encore peu explor du fait de la difficult de mettre au point une mthode de purification efficace dans ce milieu complexe.
324/381
[P238] Proteomic mapping of glyphosate-exposed human HaCaT keratinocytes using two-dimensional electrophoresis and atomic force microscopy
Cline Heu1, Graldine Lucchi1, Alexandre Berquand4, Patrick Ducoroy1, Cline Elie-Caille1, Laurence Nicod1
1 2
plateforme proteomic CLIPP, CLIPP CGFL 1 rue du Professeur Marion BP 77980 , 21019 Dijon
Laboratoire de Biologie Cellulaire EA 4267 Universit de Franche Comt, UFR SMP Place Saint Jacques, 25030 Besancon Cedex
3
Institut FEMTO-ST UMR 6174 CNRS, Universit de Franche Comt, 32 avenue de l'observatoire, 25044 Besancon Cedex
4
Brucker Nano Surfaces Division, Dynamostrasse , 19, 68165 Mannheim (Germany)
Contact: Cline Heu (heu.celine@univ-fcomte.fr)
Keywords: Systems biology, Instrumentation The epidermal differentiation and the different cell death programs need to be well orchestrated to maintain skin homeostasis and the biological barrier function. Environmental chemical aggressions can lead to a deregulation of one of these mechanisms and consequently to cutaneous pathologies. We previously showed that glyphosate, an extensively used herbicide, caused cytotoxic effects on cultured human keratinocytes, affecting their antioxidant capacities and impairing morphological and functional cell characteristics through an apoptosis phenomenon. The aim of the present study was to investigate the cytosolic and membrane proteomic profiles of human HaCaT keratinocytes in order to measure the cytoxic effects of glyphosate. Different incubation periods and glyphosate concentrations were performed on HaCaT cells, followed by two-dimensional electrophoresis in conjunction with MALDI-TOF/TOF mass spectrometry analysis. The data showed significant modifications in the expression of proteins known to be implying in intracellular oxidative stress mechanisms. AFM has been established as a versatile tool for imaging topography and measurements of mechanical properties on living cells. An original and complementary study by the QFM-based Peak Force QNM technology offers the opportunity to perform proteomic mapping on living cell membrane, using coated tip with specific antibodies. This strategy will allow us to reinforce results obtained with the classical proteomic study on living cells. This AFM technology has been already used to obtain topography images and high resolution characterization of the individual cell mechanical properties on our epidermal in vitro model. Eventually a better understanding of the glyphosate-induced deleterious mechanisms will help us in making a relevant choice of antioxidant molecules able to reverse the cellular impairments.
325/381
[P239] Etude de la bioaccumulation et de la mtabolisation de la fluoxtine dans des invertbrs benthiques par Micro-QuEChERS-NanoLC-Nano-ESI-MS/MS
Audrey Bulet 1, Robert Baudot1, Laure Wiest1, Marion Gust2, Jeanne Garric2, Ccile Cren-Oliv1
1 2
Service Central d'Analyse-UMR5280ISA, chemin du canal, 69360 Solaize Laboratoire Ecotoxicologie biologie du CEMAGREF, 3 bis quai Chauveau , 69336 Lyon
Contact: Audrey Bulet (a.bulete@sca.cnrs.fr)
Keywords: Proteins, peptides and small molecules quantification La fluoxetine est un antidpresseur largement utilis, retrouv frquemment dans les cosystmes aquatiques. Cette molcule prsente des effets diffrents sur la reproduction de deux espces gastropodes, Potamopyrgus antipodarum et Valvata piscinalis. Rcemment, elle a t mesure dans des poissons en aval des rejets d'effluents indiquant une capacit de bioaccumulation leve chez les vertbrs de la faune aquatique. Toutefois, aucune donne n'est disponible sur le devenir et le mtabolisme de mdicaments chez les invertbrs. Pour mieux comprendre la sensibilit interspcifique (espce dpendante), la bioaccumulation de la fluoxtine et sa mtabolisation in vivo en norfluoxtine sont explorer chez P. Antipodarum et V. Piscinalis. Ce type d'tude ncessite le dveloppement d'outils analytiques pour l'extraction et pour l'analyse de traces de fluoxtine et de norfluoxtine dans des matrices environnementales comme les invertbrs benthiques deau douce, gastropodes ne pesant que quelques milligrammes. La complexit et la trs faible quantit de lchantillon rendent dautant plus dlicates, aussi bien ltape dextraction que lanalyse proprement dite. En effet, les substances recherches ltat de traces dans des matrices complexes de si petite taille ncessitent la miniaturisation des techniques existantes et lutilisation de technologies de pointe : la nano chromatographie liquide couple la spectromtrie de masse en tandem (nano-LC-nano-ESI MS/MS) permet daugmenter la sensibilit des analyses, de diminuer ainsi les limites de dtection et de pouvoir rpondre ces enjeux cotoxicologiques. Ainsi, lutilisation de technologies de pointe est ncessaire pour rechercher des traces de mdicaments dans des matrices complexes comme les gastropodes. Dans ce but, nous avons tabli une stratgie analytique qui consiste en une seule extraction pour la fluoxtine et la norfluoxtine base sur la mthode QuEChERS, suivie d'une analyse par nano-LC-MS/MS. En effet, la nanochromatographie couple la spectromtrie de masse en tandem (nano-LC-nano-ESI MS/MS) augmente la sensibilit, rduit la quantit d'chantillon initial requise et constitue un bon outil pour rpondre cette question cotoxicologique. Les objectifs de cette tude sont (i) de dterminer si la fluoxtine a t bioaccumule dans les escargots aprs exposition cette dernire; (ii) d'valuer sa mtabolisation in vivo en norfluoxtine; (iii) d'valuer la variabilit interspcifique de bioaccumulation et de biotransformation de la fluoxtine, afin de mieux comprendre les diffrences de sensibilit entre P. Antipodarum et V. Piscinalis. De plus, la mthode, dveloppe lchelle dun individu permet denvisager une intgration des donnes dans une approche mtabonomique permettant de caractriser et de paramtrer la diversit des rponses biologiques obtenues.
326/381
[P240] Modifications post-traductionelles de Pin1 dans des Tauopathies

Yann Verdier1, Sabiha Eddarkaoui2, Emmanuelle Demey-Thomas1, Nicolas Sergeant2, Luc Bue2, Jolle Vinh1
1
Laboratoire de Spectromtrie de Masse et protomique USR 3149 CNRS ESPCI ParisTech, 10 rue Vauquelin, 75005 Paris
2
Centre Jean Pierre Aubert, IMPRT, Univ. Lille II, 1, Place de Verdun, 59045 Lille Cedex
Contact: Yann Verdier (yann.verdier@espci.fr)
Keywords: Systems biology, Clinical proteomics Un pr requis de la dphosphorylation des phospho-srines ou des phospho-thronines suivis de rsidus prolines est lisomrisation des liaisons peptidiques imidiques prcdant le rsidu proline. Ce mcanisme est assur par une peptidyl-prolyl cis/trans isomerase nomm Pin1. Par ces isomrisations, Pin1 rgule la fonction dun nombre croissant de cibles, dont la protine Tau associe aux microtubules, qui est hyperphosphoryl et agrge dans les neurones en dgnrescence dans la maladie dAlzheimer et les Tauopathies. En utilisant un modle de neuroblastome SY5Y exprimant de faon inductible la protine Pin1, et ces mmes cellules traites avec 125 nM acide okadaque, des analyses de western blot en 2D ont montr que la prsence de cinq isoformes de Pin1, variant principalement par leur point isolectrique. Afin didentifier les modifications post-traductionnelles responsables de ces isoformes, ces isoformes isoles de gels 2D ont t analyss par MALDI TOF, MALDI Orbitrap, et LC FT MS. Ces analyses ont permis de mettre en vidence que plus de cinq rsidus de Pin1 peuvent tre modifis, en particulier par des oxydations et des actylations N terminales. Des analyses complmentaires visant prciser la relation entre la spcificit de ces modifications et les pathologies Tau, en particulier dans la maladie dAlzheimer, constituent la suite de ces travaux.
327/381
[P241] Dveloppement dune approche mtabolomique pour le dpistage de strodes anabolisants dans lurine quine
Natali Stojiljkovic1, Patrice Garcia1, Marie-Agns Popot1, Yves Bonnaire1, Alain Paris2, Christophe Junot3, JeanClaude Tabet4
1 2 3 4
Laboratoire des Courses Hippiques (LCH), 15 rue de Paradis, 91370 Verrires le Buisson, France Met@risk INRA, 16 rue Claude Bernard, 75231 Paris Cedex 05 Commissariat l'Energie Atomique (CEA, LEMM), Centre de Saclay, Bat 136, 91191 Gif-sur-Yvette Cedex
Laboratoire de Chimie Structurale Organique et Biologique (LCSOB), UMR 76 13, Bat F72, 4 place Jussieu, 75252 Paris Cedex 05 Contact: Natali Stojiljkovic (n.stojiljkovic@lchfrance.fr)
Keywords: High mass and high resolution mass Spectrometry Les travaux par approche mtabolomique relatifs la dtection de ladministration dhormone de croissance chez le cheval et ceux concernant la dtection du clenbutrol chez les bovins ont montr lintrt de cette approche pour notre contexte de recherche. Ainsi, nous orientons notre tude vers un profilage mtabolique urinaire non cibl afin de prendre en compte lensemble des perturbations dtectes suite ladministration de strodes anabolisants, pour mettre en vidence leur utilisation chez le cheval. L'objectif est dvaluer le potentiel dune telle approche dans le dpistage de strodes anabolisants. Le stanozolol, un strode anabolisant exogne, a t choisi pour dmontrer lapplication de cette mthode une matrice biologique trs complexe qui est lurine de cheval. Afin de diffrencier une population non traite d'une population traite au stanozolol, une phase animale approprie a t ralise afin d'tudier les variations physiologiques du profil mtabolomique. Pour cela, 14 juments ont t suivies pendant prs d'un an et des chantillons d'urine ont t recueillis deux fois par mois. Ensuite, 14 juments de l'tude prcdente ont t impliques dans le protocole d'administration. Deux groupes de 5 juments ont reu un traitement chronique de stanozolol (4 fois tous les 4 jours) deux doses diffrentes, par IM. Les 4 autres juments ont reu le vhicule dadministration. Les chantillons ont t collects pendant huit mois. Pour cette tude, notre choix danalyseur sest port sur un hybride quadriple-temps de vol (micrOTOF-QII, Bruker) du fait de ses caractristiques ncessaires pour la recherche des biomarqueurs dans une approche globale (rsolution, prcision en masse, vitesse de scan, MS/MS et gamme dynamique). Lelectrospray a t choisi comme mode dionisation en positif et ngatif. Le couplage a t effectu avec un systme HPLC (Ultimate, Dionex), avec une colonne chromatographique Uptisphere C18 (100 *2,1 mm, 2,2 m) avec un temps danalyse de 25 min. La prparation des chantillons consiste en une prcipitation lactonitrile. Les conditions de traitement des donnes avec le logiciel XCMS et les analyses statistiques multivaries avec le logiciel SIMCA P (Umetrics) ont aussi t dfinies. Les rsultats qui en dcoulent ont permis de mettre en vidence une modification du mtabolome des chevaux aprs traitement au stanozolol. Les facteurs physiologiques qui influent fortement le mtabolome ont t minimiss en choisissant des chevaux du mme sexe, du mme ge et de mme race, mais on a observ dautres facteurs (individuels, saisonniers et environnementaux) qui modifient de faon significative le mtabolome des chevaux. Ces rsultats semblent montrer que certains de ces facteurs peuvent masquer linformation lie aux perturbations mtaboliques entraines par ladministration de substances prohibes et quils sont donc prendre en considration.
328/381
[P242] Spciation des mtaux et des mtallodes dans des plantes modles par lassociation des couplages LC ICP-MS et LC ESI-LTQ Orbitrap MS
Laurent Ouerdane1, Paulina Flis1, Ryszard Lobinski1
1
Laboratoire de Chimie Analytique Bio-inorganique et Environnement, UMR5254 Universit de Pau et des Pays de l'Adour / CNRS, 2 avenue du prsident Angot, 64000 Pau, France Contact: Laurent Ouerdane (laurent.ouerdane@univ-pau.fr)
Keywords: Organic and inorganic mass spectrometry, Separative analysis methods Habituellement complexs ou lis des molcules organiques, de nombreux mtaux et mtallodes jouent un rle essentiel dans le mtabolisme des plantes, en particulier pour lactivit enzymatique et la prservation des structures de la cellule. Dun autre ct, un excs de mtaux peut provoquer une toxicit importante en se substituant aux mtaux essentiels prsents dans les enzymes ou par simple dommage oxydant. Sadaptant aux conditions des sols, les plantes ont dvelopp des mcanismes pour augmenter ou diminuer slectivement lincorporation et laccumulation des mtaux dans leurs tissus permettant au fragile quilibre entre essentialit et toxicit dtre maintenue en produisant des mtabolites complexant les mtaux, des protines membranaires impliques dans le transport de mtaux et leur squestration dans des compartiments pour leur stockage. Dans les plantes, les mtabolites impliqus dans le transport et le stockage des mtaux essentiels nont que trs peu t tudis jusqu maintenant. De toute vidence, la faible concentration de ces complexes mtalliques et leur labilit variable durant la prparation des chantillons et les sparations chromatographiques sont les principales raisons ce manque dinformations. Pour permettre des analyses de spciation, les rsultats obtenus soit par couplage LC ICP MS cellule de collision (information lmentaire sensible et non interfre) ou LC-ESI LTQ Orbitrap MS (information molculaire sensible et haute rsolution). Le travail didentification fut ainsi simplifi y compris avec des chantillons non purifis et faiblement concentrs en mtaux, ce qui permit dobtenir des informations plus significatives sur la forme relle des espces mtalliques dans les chantillons originaux. De nombreuses espces mtalliques (une cinquantaine) purent ainsi tre identifies pour la premire fois dans des plantes modles Pisum sativum (le petit pois), Arabidopsis halleri et des crales et ceci pour de nombreux mtaux ou mtallodes (Fe, Zn, Cu, Se, Mo, Ca, Mg, Ni, Co, Mn).
329/381
[P243] Dveloppement dun spectromtre FTICR transportable associ une source dionisation ambiante pour la dtection de composs peu volatils
Jol Lemaire1, Clotilde Le Vot1, Hlne Mestdagh1, Pierre Boissel2, Grard Mauclaire2, Michel Hninger2
1 2
Laboratoire de Chimie Physique, Btiment 350 Universit Paris Sud, 91405 Orsay, France AlyXan, Btiment 207B Centre Universitaire d'Orsay, 91405 Orsay, France
Contact: Jol Lemaire (joel.lemaire@u-psud.fr)
Keywords: Instrumentation, Organic and inorganic mass spectrometry La spectromtrie de masse FTICR repose sur lutilisation de champs magntiques intenses et homognes. Ceux-ci sont aujourdhui souvent raliss avec des aimants supra conducteurs qui permettent de raliser des champs levs (jusqu 15 Tesla disponibles commercialement) et avec une trs grande homognit (variation relative de quelques ppm dans le volume de la cellule). Mais ils ont comme dsavantage un cot lev tant lachat qu lentretien (en raison des fluides cryogniques ncessaires leur fonctionnement) et un poids lev ce qui en fait des instruments certes trs performants mais ncessairement fixes dans un laboratoire. Nous nous sommes orients vers la ralisation dinstruments bass sur des aimants permanents structurs, qui permettent dobtenir des champs compris entre 1 et 1.5 Tesla avec des homognits relatives de lordre de 10-3 dans le volume de la cellule. Ces instruments ne cherchent pas concurrencer les FTICR supraconducteurs dans les domaines de la protomique ou de la ptrolomique o ceux-ci excellent. Nous visons la dtection de molcules plus petites, les composs organiques volatils, en mettant profit la haute rsolution pour distinguer les molcules diffrant par leur formule chimique mme lorsquelles ont des masses nominales identiques. Ceci est essentiel pour les applications dans le domaine de la surveillance environnementale ou dans celui de la scurit car des molcules toxiques et inoffensives peuvent avoir la mme masse nominale. Nous avons dj dmontr les performances qui peuvent tre obtenues en utilisant des structures de Halbach (cylindrique gnrant un champ transverse) et en mettant en uvre lionisation chimique partir de diffrents prcurseurs dans la cellule ICR. Les limites de dtection obtenues sont toutefois de lordre de la ppm et les composs trs peu volatils prsents ltat de trace peuvent se perdre dans les lignes dintroduction. Cest pourquoi nous avons dvelopp une nouvelle configuration compacte base sur un aimant gnrant un champ coaxial avec laxe principal de la structure magntique cylindrique. Avec cette configuration les ions peuvent tre forms lextrieur du champ magntique. Nous avons choisi de les produire pression atmosphrique, dans des conditions ambiantes, par raction partir des atomes excits mtastables gnrs dans une source dcharge. Les ions sont transfrs de la pression atmosphrique jusqu 10-8 mbar dans la cellule ICR grce une introduction pulse et lutilisation de deux hexapoles, le premier pour mettre les ions en paquet et le deuxime pour les guider vers lenceinte contenant la cellule ICR. Les premiers rsultats sur diverses molcules prsentes ltat de trace dans lair seront prsents.
330/381
[P244] Proteomic dissection of the Arabidopsis thaliana vacuolar proteome. New insights into the composition and molecular mass of protein complexes of plant vacuole.
Nolwenn Jarno1, Sylvie Kieffer-Jaquinod1, Florent Villiers1, Jrme Garin1, Christophe Bruley1, Michel Jaquinod1, Jacques Bourguignon1
1
Laboratoire d'tude la Dynamique des Protome, BGE, iRTSV, 17 rue des Martyrs, 38000 Grenoble, France
Contact: Nolwenn Jarno (nolwenn.jarno@gmail.com)
Keywords: Signaling, interactomics and post-translational modifications, Systems biology, Separative analysis methods The vacuole is a multifunctional organelle characteristic of plant cells, playing a central role in cellular physiologies. The vacuole allows the storage of many metabolites, ensures care of turgor pressure, pH regulation and ionic homeostasis via the storage or release of solutes and ions through transporters present at the vacuolar membrane, the tonoplast. Among functions, the vacuole has a key role in cellular protection by neutralizing the compounds that could interfere with the normal metabolic pathways processes. It allows sequestration or degradation of xenobiotics and toxic compounds. Cellular detoxification mainly dependents on the vacuole, but the mechanisms of transport and storage of toxics in the vacuole is still unclear. To identify proteins involved in these mechanisms we have developed a procedure to prepare intact highly purified vacuoles. We used protoplasts isolated from Arabidopsis thaliana cell cultures. Based on the specific activity of the vacuolar marker -mannosidase, preparations showed the necessary degree of purity for proteomic study. We were interesting in the characterization of the soluble and membrane vacuolar fractions. Analysis of the vacuolar sap has identified over 500 proteins, 70% of them are enzymes. Behinds the 950 tonoplastic proteins described around 130 transporters were characterized. To go into insight the vacuolar proteome fine location of proteins were carried out by the use of shave-and-conquer concept and quantification based on spectral counting. We reassessed acute vacuolar protein location (ie full membrane or associated to the external or internal membrane) and vacuole/extravacuole distribution of some soluble protease. Using biochemical and proteomics approaches, we present the first evidence of active proteasome / subtilase degradative pathway associated to the plant tonoplaste. Then to look into the supra molecular proteins organization blue-native polyacrylamide gel electrophoresis (BN/SDS-PAGE) was used. We applied this dividing method in native condition to reveal the presence of putative complexes in the soluble and tonoplastic vacuolar proteome such as complexes of ubiquitin specific peptidase and proteasome system. Altogether, the present proteomic work constitutes the basis to study the dynamics of the vacuolar proteome in response to several stresses.
331/381
[P245] La Satisfaction client sur les plateformes danalyses protomiques

Cline Fleury1, Myriam Ferro1, Yohann Cout1, Christophe Bruley1
1
CEA, iRTSV, Laboratoire dEtude de la Dynamique des Protomes, Grenoble, F-38054, France ; INSERM, U880, Grenoble, F-38054, France ; Universit Joseph Fourier, Grenoble, F-38054, France., 17 rue des Martyrs, 38054 Grenoble Contact: Cline Fleury (celine.fleury@cea.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics Raliser des demandes danalyse en satisfaisant aux exigences initiales du client public ou priv dans les dlais impartis est une prrogative de chaque plateforme danalyse, gage dune certaine expertise et dutilit pour la collectivit scientifique. Mais comment mesurer cette satisfaction et la perception qua le client de la plateforme ? Quels sont les outils pour amliorer et augmenter la performance du service ? La norme ISO 9001 version 2008 en fait une obligation de part ses exigences au chapitre 8.2.1. Dans le cadre de la mise en uvre dune dmarche qualit au sein du laboratoire EDyP, nous montrerons quelques exemples de moyens de mesure de la satisfaction client sur la plateforme EDyP-Service (CEA/Grenoble) et les actions damlioration mise en place suite aux remarques des clients.
332/381
[P246] Comparaison des sources dionisation APCI et ESI pour le suivi dadditifs utiliss dans la formulation de conditionnements plastiques alimentaires et pharmaceutiques
Charlne Pouech1, Robert Baudot1, Laure Wiest1, Didier Lonard2, Ccile Cren-Oliv1
1 2
ISA, UMR 5280, Service Central dAnalyse du CNRS, Echangeur de Solaize, 69360 Solaize, France
ISA, L.S.A - Laboratoire des Sciences Analytiques, UMR 5180, Btiment CPE, Universit Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France Contact: Charlne Pouech (c.pouech@sca.cnrs.fr)
Keywords: Organic and inorganic mass spectrometry Au cours de ces dernires annes, l'engouement non ngligeable pour les emballages base de polymres sexplique par leur caractre mallable, incassable, solide, lger, recyclable, transformable et leur remarquable rapport qualit/prix. Lindustrie polymre est dsormais en mesure de produire des emballages plastiques de meilleure qualit et rpondant aux exigences requises dans les secteurs alimentaires et pharmaceutiques grce notamment lincorporation de substances, appeles additifs, des polymres naturels ou synthtiques. Cependant, linertie dun emballage est rarement totale vis--vis du contenu. Diffrents phnomnes dinteractions peuvent effectivement se produire entre le contenant et le contenu. La migration, aussi appel le relargage, correspond au phnomne le plus inquitant pour la sant du consommateur. En effet, la migration des additifs, prsents initialement dans lemballage, vers laliment ou la solution pharmaceutique peut alors altrer la qualit du produit, ou le rendre toxique. Les industries alimentaires et pharmaceutiques se soucient alors de ce phnomne, et le matrisent. Elles imposent ainsi dans leurs domaines respectifs le nombre dadditifs, la nature des additifs ainsi que les teneurs maximales autorises incorporer dans les produits finis. Une mthode de rfrence en HPLC-UV existe dores et dj pour identifier et quantifier le relargage dun certain nombre dadditifs chelle industrielle. Cependant, la prsence gnralement ltat de traces d'additifs dans les produits contenants ncessite le dveloppement de mthodes beaucoup plus sensibles et spcifiques. Dans ce contexte, nous avons choisi de dvelopper une mthode multi rsidue en HPLC-MS/MS. Nanmoins, la difficult de lanalyse des additifs rside dans la grande varit de leurs proprits physicochimiques telles que leur masse molculaire, leur polarit, leur thermolabilit et leur solubilit dans les solvants organiques. Une comparaison des sources dionisation APCI et ESI simpose alors. Ltude technique a pour but de dterminer le mode dionisation optimal, mode pour lequel les limites de dtection et de quantification seront les plus faibles pour le suivi de la migration dadditifs autoriss dans les deux domaines dapplication et de certains de leurs produits de dgradation. Loptimisation de cette mthodologie analytique permettra de documenter la prsence d'additifs dans des emballages aussi bien alimentaires que pharmaceutiques.
333/381
[P247] Analyse multi-rsidue dhormones libres, conjugues et de perturbateurs endocriniens dans les organes sexuels de rat par QuEChERS et LC-MS/MS
Mikal Tournier1, Charlne Pouech1, Laure Wiest1, Florent Lafay1, Ccile Cren-Oliv1
1
Service Central d'Analyse (SCA), UMR5280 CNRS, Chemin du Canal, Echangeur de Solaize, 69360 Solaize, France Contact: Mikal Tournier (m.tournier@sca.cnrs.fr)
Keywords: Proteins, peptides and small molecules quantification Apparue la fin du XXme sicle, la notion de Perturbateurs Endocriniens dsigne un compos xnobiotique proprit hormono-mimtique. Il sagit en fait dune substance exogne pouvant avoir un impact sur lquilibre hormonal d'organisme et qui a des effets nocifs sur la sant de cet organisme et de ses descendants. Lactivit perturbatrice endocrinienne est reconnue lorsque la relation de causalit entre lexposition une substance souponne tre perturbatrice endocrinienne et les effets nfastes sur la sant ont t mis en vidence et prouvs. Le Bisphnol A, la Vinclozoline, lAtrazine et le Mthoxychlore sont ainsi suspects dtre des perturbateurs endocriniens. Afin dtudier limpact de ces molcules sur le systme hormonal et la diffrenciation sexuelle, le suivi de la balance andrognes/estrognes dans les organes reproducteurs (ovaires et testicules), par exemple, de rats exposs ces contaminants simpose. Or, si des mthodes danalyse ont t dcrites pour la quantification de perturbateurs endocriniens dans diffrentes matrices environnementales, la littrature se rvle trs pauvre en ce qui concerne les matrices biologiques et plus particulirement les organes reproducteurs. Les dtections et quantifications de contaminants organiques dans ce type de matrices sont de vritables dfis analytiques compte tenu de la diversit des substances dangereuses recherches, de leur prsence gnralement ltat de traces, de la complexit des matrices biologiques qui rend dlicate ltape dextraction. Dans ce contexte, il est ncessaire de dvelopper une mthode danalyse permettant d'extraire, de dtecter et de quantifier la fois les perturbateurs endocriniens et les hormones permettant de suivre la balance andrognes/estrognes induisant respectivement les caractres sexuels masculins et fminins. Les andrognes recherchs sont : la Testostrone (T) et lAndrostnedione (A). Les estrognes recherchs sont : lEstrone (E1), lEstradiol (E2) et leurs mtabolites sulfo- et glucuro-conjugus associs (E1S et E2S ; E1G et E2G). Ltape cl de ces analyses reste la prparation des chantillons, aussi avons nous optimis cette tape par l'utilisation d'une technique innovante QuEChERS. La stratgie analytique adopte ensuite est base sur lanalyse en LC-MS/MS afin dobtenir des limites de quantification compatibles avec les tudes engages : de 20 60 pg/g. Loptimisation de cette mthodologie analytique a permis de documenter l'effet de perturbateurs endocriniens sur la balance hormonale de mammifres.
334/381
[P248] Differential proteomics for the evaluation of the effect of environmental culture conditions on the protein expression of somatic embryos from Pinus pinaster
Martine Beaufour1, Alexandre Morel2, Caroline Teyssier 2, Luc Harvenght3, Jean-Franois Trontin3, Marie-Anne Lelu-Walter2, Philippe Label2, Martine Cadene1
1 2 3
Centre de Biophysique Molculaire, CNRS UPR4301, rue Charles Sadron, 45071 Orlans cedex 2, France UAGPF, INRA , 2163, avenue de la Pomme de Pin - CS 40001 Ardon, 45075 Orlans Cedex 2 France LBT, FCBA, Nangis Domaine de lEtano, 77370 Nangis France
Contact: Martine Beaufour (martine.beaufour@cnrs-orleans.fr)
Keywords: Systems biology Context: In order to get insights into the physiological mechanism underlying the development of somatic embryos of the maritime pine (Pinus Pinaster), experiments at different stages of embryos maturation and different water availability conditions were undertaken to identify protein markers. To our knowledge, this is the first proteomic investigation on somatic embryos of Pinus pinaster. We will highlight in this presentation the effect of environmental culture conditions on the protein profile. Method: Embryo cultures and 2D gels of the corresponding protein extracts were processed by INRAs team. The maturation of the embryos was realised in presence of either low or high gellan gum concentrations.The two cultures were harvested at one week of maturation. Following numerical analysis of 2D gels, only the statistically significant spots were excised from the gel. These spots were subjected to enzymatic proteolysis followed by nanoLC-ESI-IT (CID) mass spectrometry analysis and database searching. As we are dealing with an unsequenced organism, identifications were performed with nucleotide databases based on expressed sequence tags (EST) or on tentative consensus sequences (TC) and protein databases. Results: More than 1428 proteins were reproducibly detected on each gel. At 1 week of maturation, the abundances of 82 spots detected in analyses of embryos matured at the two gellan gum concentrations significantly differed. Among them, the corresponding proteins of 54 spots were reliably identified by LC-MS/MS, and were found to be mainly involved in Carbohydrate metabolism and Genetic information processing according to KEGG taxonomy. The database searching methods will also be discussed. Outlook: This work will be expanded to other factors influencing embryo development with the aim to facilitate species propagation. The results will be combined with the evaluation of gene expression and sequencing of corresponding RNAs. Acknowledgement: This work was funded by the Region Centre Embryome project (n33000111).
335/381
[P249] Study of the chloroplast proteome during the ripening of tomato fruit.
Isabel Egea1, Cristina Barsan1, David Bouyssi3, Elie Maza1, Carole Pichereaux4, Michel Rossignol4, Mohamed Zouine1, Wanping Bian1, Alain Latche1, Jean-Claude Pech1
1
Universit de Toulouse, INP-ENSA Toulouse, gnomique et Biotechnologie des Fruits, Avenue de l'Agrobiopole, 31326 Castanet-Tolosan
2 3 4
INRA, Gnomique et Biotechnologie des Fruits, Chemin de Borde rouge, 31326 Castanet-Tolosan Universit de Toulouse, UPS, IPBS, Plateforme protomique, 205, route de Narbonne, 31077 Toulouse
Fdration de Recherches 3450, Agrobiosciences, Interactions et Biodiversit, 24 chemin de Borde Rouge, 31326 Castanet-Tolosan Contact: Jean-Claude Pech (pech@ensat.fr)
Keywords: Systems biology, Proteins, peptides and small molecules quantification , Bioinformatics et biostatistics dedicated to proteomics Fruit ripening is accompanied by macroscopic events such as changes in color, texture and flavors that render the fruit attractive to consumers. At the cellular level, alterations are particularly evident at the headquarters of the photosynthetic organelle, the chloroplast. Throughout the maturation process, it undergoes molecular rearrangements that result in morphological changes.. In particular, a loss of chlorophylls and a re-ordering of the plastid structure occur that participate in the conversion of a chloroplast into a chromoplast. In the present study we have studied the changes in the proteome during the transition from chloroplast to chromoplast in ripening tomato fruit. Three developmental stages have been selected according to the color changes: an early stage, mentioned as "mature-green", an intermediate stage named "breaker" corresponding to the initiation of red color and finally a "red" stage corresponding to the fully ripe fruit. For each stage, plastids were purified by density gradient separation. Total proteins were extracted, separated by one-dimensional electrophoresis, digested with trypsin and the peptides were finally analyzed by chromatography (nano-HPLC, Dionex) coupled to a mass spectrometer (Orbitrap). The identification of proteins using the MASCOT tool was facilitated by the recent availability of genome annotated database of tomato. For quantification by label-free ("Spectral counting") and its statistical validation, 3 biological replicates were performed for each stage. The whole results corresponding to nearly 3000 proteins was sorted and compared using the "MapMan" and "Proteincenter tools. Proteins were classified into metabolic functions and quantitative changes were evaluated during the chloroplast_to chromoplast transition. Besides confirming the breakdown of the photosynthetic system and the increase in the carotenoid biosynthetic pathway, our data indicate that several functions present in the chloroplast persist in the chromoplast while others undergo profound re-orientations. These data will now allow a better description of the molecular processes involved in the differentiation of this organelle and will provide new molecular targets for understanding the regulation of fruit ripening and development of sensory quality.
336/381
[P250] Assay development on the NanoPro Platform: 4E-BP1 and 4E-BP2.

Uyen Nguyen1, Annegret Bodge1
1
Cell Biosciences, 3040 Oakmead Village Drive, CA 95051 Santa Clara - USA
Contact: Uyen Nguyen (tsalomon@cellbiosciences.com)
Keywords: Signaling, interactomics and post-translational modifications, Clinical proteomics, Instrumentation Assay development on the NanoPro Platform: 4E-BP1 and 4E-BP2 Uyen Nguyen Research Associate, Annegret Bodge Director R&D Cell Biosciences Abstract : We present the development of novel immunoassays for the translational repressor proteins 4E-BP1 and 4E-BP2 using NanoPro technology. Both the PI3 kinase / Akt pathway and FRAP / mTOR kinase pathway regulate 4E-BP1 activity, making 4E-BP1 a focal point for these two important signaling pathways. 4E-BP2 regulation is poorly understood, partially due to the lack of specific anti-phospho 4E-BP2 antibodies. Our assay, developed on the Cell Biosciences NanoPro platform, enables detailed differential investigation of 4E-BP1 and 4E-BP2 phosphorylation and signal transduction. Link: www.cellbiosciences.com
337/381
[P251] Proteomic identification of calumenin as a G551D - CFTR associated protein.

Mehdi Taiya1, Pascal Trouve2, Ling Teng2, Nathalie Benz2, Mathieu Kerbiriou2, Olivier Mignen2, Claude Ferec4
1
Service commun de spectrometrie de masse, Universite de Bretagne Occidentale, 06, avenue Victor Le Gorgeu, 29200 Brest
2 3
Inserm, U613, 46, rue Felix Le Dantec , 29218 Brest
Universite de Bretagne Occidentale, Faculte de Medecine et des sciences de la sante, 22, rue Camille Desmoulins, 29200 Brest
4 5
Etablissement Francais du Sang - Bretagne, 46, rue Felix Le Dantec , 29200 Brest C.H.U. Brest, Hopital Morvan, Laboratoire de Genetique Moleculaire, 02, avenue Foch , 29200 Brest
Contact: Mehdi Taiya (mehdi.taiya@univ-brest.fr)
Keywords: Systems biology Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population, and is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To date, over 1700 mutations have been identified in the CFTR gene. Among these mutations, the CF-causing missense mutation G551D-CFTR (approx. 5% of cases) exhibits normal expression on the cell surface but it is associated with severe disease due to its altered channel activation. The aim of the present study was to identify specific interacting proteins of G551D-CFTR. Membrane proteins from Wt-CFTR and G551D-CFTR expressing cells were extracted and resolved by 2D-gel electrophoresis (2-DE). Mass Spectrometry revealed that calumenin was only present in the proteins extracted from G551D-CFTR expressing cells. Coimmunoprecipitations showed that calumenin is linked to Wt- and to G551D-CFTR. The amount of bound calumenin was higher in G551D-CFTR protein complex than in wt-CFTR complex. Nevertheless, its basal expression was not modified in G551D-CFTR expressing cells when compared to Wt-CFTR. Calumenin associated proteins were resolved by 2-DE and the spots were analyzed by MS. The results indicated that calumenin is bound to chaperons, cytoskeleton proteins and Grp78/Bip which is involved in the Unfolded Protein Response (UPR). Therefore, we showed a calumenin-CFTR interaction and we suggest that calumenin maybe involved in the G551D-CFTR maturation and trafficking pathway. Finally, we suggest that UPR may be triggered independently of the retention of G551D-CFTR in the ER. Link: http://www.genetic-brest.fr/
338/381
[P252] Etude de la symbiose chimiotrophe chez Bathymodiolus azoricus: une approche mtaprotomique
Cla BAUVAIS1, Isabelle BOUTET2, Arnaud TANGUY2, Emmanuelle DEMEY3, Jolle VINH3, Jean MARY1
1
Ecophysiologie des invertbrs marins en milieu extrmes, UMR CNRS - UPMC 7144, Place George Teissier, 29 282 Roscoff, France
2
Gntique de l'adaptation aux milieux extrmes, UMR CNRS - UPMC 7144, Place George Teissier, 29 282 Roscoff, France
3
Spectromtrie de masse biologique et protomique, SMBP CNRS USR 3149, 10 rue Vauquelin, 75005 Paris, France
4
Univ Paris Diderot, Sorbonne Paris Cit, UFR des Sciences du vivant, 5 rue Thomas Mann, 75013 Paris
Contact: Jean MARY (jmary@sb-roscoff.fr)
Keywords: Systems biology, Systems biology Les sources hydrothermales, situes sur les dorsales ocaniques, prsentent des conditions environnementales a priori hostiles au dveloppement de la vie (tempratures leves, faibles taux doxygne, pression leve, importantes concentrations en mtaux et composs rduits, absence de lumire). Ces sites hbergent cependant une faune endmique trs abondante. Ltude propose ici se concentrera sur lespce Bathymodiolus azoricus qui est un bivalve colonisateur des sites de la ride mdio-Atlantique. B. azoricus a dvelopp des adaptations physiologiques uniques pour faire face aux conditions hydrothermales. Par exemple, la mise en place dune symbiose non obligatoire avec des gamma-protobactries thiotrophes (SOX) et mthanotrophes (MOX), au sein de cellules spcialises du tissu branchial, les bactriocytes. Ces bactries permettent leur hte de coloniser des environnements riches en sulfure et en mthane. La production primaire des symbiotes partir de ces composs rduits assure lapport nutritif ncessaire la survie de lhte. Une approche de mtaprotomique compare (lectrophorse bidimentionnelle et spectromtrie de masse, MALDI-TOF/TOF) sur des tissus branchiaux de B. azoricus collects sur les sites Menez Gwen et Rainbow aux paramtres physicochimiques bien dfinis (concentration en sulfure et mthane, pH, temprature) a t mene. Ceci a permis didentifier des protines de lhte et des symbiotes diffrentiellement exprimes en fonction des sites dtude et de la charge symbiotique des individus, dans la perspective de mieux comprendre la mise en place de la symbiose, la rgulation de la charge symbiotique et ses consquences au niveau protique chez B. azoricus.
339/381
[P253] Etude de la distribution spcifique de sels de benzalkonium par imagerie par spectromtrie de masse : Application la pharmacocintique oculaire chez le lapin.
Raphael Legouffe1, Nicolas Desbenoit2, Grgory Hamm1, Christophe Baudouin2, Alain Brunelle3, Isabelle Fournier4, Olivier Laprvote3, Maxence Wisztorski4, Franoise Brignole-Baudouin2, Jonathan Stauber1
1 2 3
ImaBiotech, Campus Cit Scientifique, 59655 Villeneuve dAscq, France Institut de la Vision, INSERM, UMR_S968, 17 rue Moreau, 75012 Paris, France
Institut de Chimie des Substances Naturelles, Centre de recherche de Gif, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
4
Laboratoire de Spectromtrie de Masse Biologique Fondamentale et Applique (FABMS), EA 4550, Universit de Lille, Universit de Lille, 59655 Villeneuve dAscq, France
5
Chimie Toxicologie Analytique et Cellulaire, EA 4463, Facult des Sciences Pharmaceutiques et Biologiques, Universit Paris Descartes, 4 Avenue de lObservatoire, 75006 Paris, France Contact: Grgory Hamm (hamm.gregory@imabiotech.com) Keywords: Imaging mass spectrometry Les chlorures de benzalkonium (BAK) sont les conservateurs les plus largement utiliss dans les collyres, prsents gnralement sous forme de sel de benzododecinium (C12) et myristalkonium (C14). Ils permettent damliorer la pntration des composs actifs. Cependant, certaines tudes ont report un possible effet toxique de ces derniers au niveau de la surface oculaire et plus particulirement dans le cadre de traitements de longue dure comme celui contre le glaucome [1]. Ainsi, limagerie par spectromtrie de masse est utilise afin de caractriser la distribution spatiale des BAKs et valuer leur impact physiologique au niveau molculaire. Cette tude a t ralise sur des yeux de lapin instills avec des solutions de BAKs pendant diffrents temps de cintique (1 ou 5 mois) et nous as permis de dtecter deux types dions m/z 304.32 et m/z 332.36 correspondant respectivement au BAK C12 et C14. Une approche multi-technique a t choisie afin de mener ces travaux utilisant la spectromtrie de masse MALDI-TOF, le TOF-SIMS [2] ou encore le MALDI-Orbitrap. Cela a permis de faciliter la dtection dun large panel de composs (petites molcules, lipides [2], peptides, etc) et daccder aux plus hautes rsolutions spectrale et latrale. Cette prsentation sera consacre plus prcisment aux rsultats dimagerie MALDI-TOFMS. Nous discuterons de la localisation spcifique et de laccumulation des BAKs dans certaines zones histologiques dintrt (corne, angle iridocornen, conjonctive, limbe,) connues pour tre des rgions inflammatoires de lil. Lapport de la haute rsolution latrale sera dmontr sur deux rgions de lil particulirement importantes que sont le trabculum et le nerf optique. Nous pointerons galement une diffrence fine de comportement des deux types de BAK suivant leur structure. De plus, leur prsence sera valide par des tudes structurales par spectromtrie de masse en tandem ou assimiles. Par exemple, le mode FAST-SRM permettra de suivre un fragment spcifique dun compos, dans notre cas, m/z 212.42 (fragment du BAK C12) et ainsi valider sa distribution au niveau des structures oculaires prcdemment cites. Des expriences croises ont galement t menes entre deux laboratoires utilisant diffrents spectromtres de masse MALDI-TOF et modes de dposition de matrice afin de valider ces donnes. Limagerie par spectromtrie de masse apparait donc comme un outil de choix pour ltude de la distribution de composs connus pour avoir des effets dltres et peut tre utile dans le cadre dtude prclinique dans les domaines pharmacologique et toxicologique. 1 Baudouin, C., Detrimental effect of preservatives in eyedrops: implications for the treatment of glaucoma. 2008, Blackwell Publishing Ltd. p. 716-726. 2 Desbenoit, N., SMAP 2011, Avignon
340/381
[P254] Biocontrol proteomics:Implication of the pentoses phosphates pathway in the antagonist effect of Pichia anomala against Botrytis cinerea on apple.
Anthony Kwasiborski1, Jenny Renaut2, Philippe Lepoivre1, M. Hassam Jijakli1
1 2
Unit of plant pathology, GxABT, University of Lige, rue des dports, 2, 5030 Gembloux, Belgium
Department of Environment and Agro-Biotechnology, CRP Gabriel Lippmann, 41, rue du Brill, 4422 Belvaux, Luxembourg Contact: Anthony Kwasiborski (akwasiborski@ulg.ac.be)
Keywords: Systems biology The growing interest of the consumers for the wholesome food and the protection of the environment as well as the development of resistant pathogens to pesticides, stimulate the interest of growers to apply biological control methods. Pichia anomala strain K was previously identified as an efficient biocontrol agent of the main apple pathogens, Botrytis cinerea and Penicillum expansum. Further study demonstrated the complexicity of the mode of action of P. anomala against B. cinerea. A cDNA-AFLP and gene disruption study revealed implication of exo-1,3-glucanases in the mode of action of P. anomala strain Kh6 (a haploid form of P. anomala strain K displaying the same biocontrol properties). However, these studies suggested also implication of other factors. The present study aims to increase our knowledge of the mode of action of P. anomala strain Kh6 against B. cinerea using an in situ approach allowing the triple interaction, host/pathogen/antagonist and the proteomic tool allowing to study the ultime expression of the genome without a priori. One 50mm wound per apple were covered by a membrane and inoculated by a P. anomala suspension then by B. cinerea or not. Samples were collected during the exponential and stationary phase to identify the early and later responses to the presence of B. cinerea. After extraction, proteins were separated on 2-D gels. Spots influenced by the presence of B. cinerea in exponential and stationary phases were identified by MALDI-ToF. One hundred five and sixty spots of proteins were influenced by the presence of B. cinerea in exponential and stationary phases respectively. In exponential phase, P. anomala Kh6 in absence of B. cinerea uses mainly the glycolysis pathway, whereas in presence of pathogen, it orientates its energetic metabolism to the oxidative phosphorylation and sets up the pentose phosphate pathway. Thanks to this new orientation, P. anomala Kh6 probably obtains energy and nucleic acids allowing to colonize the wound as fast as in absence of B. cinerea and prevents the use of nutrients by the pathogen. In stationary phase, no differences in the P. anomala Kh6 energetic metabolism, in absence and in presence of B. cinerea were observed. During that phase, P. anomala Kh6 seems to use the alcoholic fermentation in order to face the nutrients impoverishment of the substrate.
341/381
[P255] Urinary exosomes : improved method for clinical applications

Ida Chiara Guerrera1, Matthieu Bourderioux2, Cerina Chhuon1, Diane-Lore Vieu2, Gabrielle Planelles2, Bernard Escudier3, Arnaud Mejean4, Thao Nguyen-Khoa2, Aleksander Edelman2
1 2 3 4
Proteomic Platform Necker, 156 rue de Vaugirard, 75015 Paris, France U845, INSERM, 156 rue de Vaugirard, 75015 Paris, France Institut Gustave Roussy, 114 rue Edouard Vaillant, 94508 Villejuif, France Hpital europen Georges Pompidou, 20 rue Leblanc, 75015 Paris, France
Contact: Cerina Chhuon (cerina.chhuon@inserm.fr)
Keywords: Clinical proteomics, Separative analysis methods Urinary exosomes are microvescicules excreted into urines from epithelial cells in urinary tract. Exosomes are a useful, non-invasive source of biomarkers in many diseases including renal cell carcinoma (RCC). Many challenges have been encountered while studying exosomal proteins. Among them, the presence of the most abundant soluble protein in urine, Tamm Horsfall Protein (THP). THP forms large filaments that may entrap exosomes and hamper the identification of less abundant proteins. The aim of present study was to establish a robust and rapid protocol for preparation of exosomes by selectively excluding the THP from the sample of exosomal proteins. First urinary miction were collected in sterile container containing antiprotease. Next, to obtain a preparation enriched in exosomes, the samples were treated according to the reference protocol with slight modifications. Subsequently, the samples were fractionated using OFF-GEL to separate THP from the rest of the proteins. Fractions obtained were separately digested and analysed by nanoHPLC-LTQ Orbitrap, maximizing the number of identification in the minimum time of analysis. THP protein was successfully removed. In the remained samples, we have identified similar number of proteins as reported by others (1171 vs 1132). The protocol was shorter and we have significantly reduced the MS analysis time. We have identified for the first time proteins that have been previously proposed as markers of a nephritic syndrome or markers of RCC. Furthermore, our method allowed to discriminate the intracellular membrane anchored THP and the secreted THP. We have established a protocol for the analysis of urinary exosomes which is suitable for screening urinary samples from patients.
342/381
[P256] Couplage de techniques analytiques la spectromtrie de masse pour lidentification de peptides en mlanges complexes
Christelle Harscoat-Schiavo1, Cdric Paris2, Xavier Framboisier1, Evelyne Ronat-Heit1, Ivan Marc1
1
Laboratoire Ractions et Gnie des Procds U.P.R. C.N.R.S. 3349, I.N.P.L., Nancy universit, Plateforme Sciences de la Vie et Sant, 13, rue du bois de la Champelle, 54500 Vanduvre-ls-Nancy
2
Laboratoire d'Ingnierie des Biomolcules, ENSAIA, I.N.P.L., Nancy universit, 2, avenue de la Fort de Haye, 54505 Vanduvre-ls-Nancy Cedex Contact: Xavier Framboisier (xavier.framboisier@ensic.inpl-nancy.fr)
Keywords: Separative analysis methods Lhydrolyse enzymatique de protines vgtales conduit un mlange complexe de peptides de tailles et proprits varies. Les petits peptides, < 1000 g/mol, prsentent un grand intrt pour les industries alimentaires, pharmaceutiques par leurs proprits physico-chimiques et leurs bioactivits potentielles. Il est ncessaire de mettre en place des stratgies rationnelles de sparation afin dadapter les tapes de purification, de caractrisation et didentification en fonction des peptides ou fractions peptidiques dintrt, en vue de leur utilisation. Le squenage est la mthode la plus prcise, mais nest pas adapte aux mlanges complexes puisquelle requiert lisolement pralable des molcules. Plusieurs techniques analytiques ont t dveloppes afin de sparer les peptides selon leurs proprits de charge, dhydrophobie et dhydrophilie. Ces sparations ont t modlises afin de caractriser et de prdire les proprits physico-chimiques dun peptide partir de son lution. La spectromtrie de masse utilise ici en couplage avec la chromatographie liquide (colonne C18 ou HILIC) comporte deux analyseurs qui travaillent en srie et/ou en parallle. Il sagit dune trappe ionique linaire (LTQ) qui permet de raliser des tudes par filiation du peptide concern c'est--dire de fragmenter n fois lion parent de la molcule dintrt, cassure partir du COOH terminal, et dune trappe ionique lectrostatique (Orbitrap) qui permet laccs la masse exacte des composs avec une rsolution maximale de 100.000 pour une masse de 400 uma. Le Laboratoire a mis au point un logiciel daide lidentification de petits peptides en mlanges complexes, dont lobjectif est de prdire au mieux leur composition lmentaire en acides amins. Il sappuie sur les informations obtenues par combinaison des techniques de sparation et leur couplage la spectromtrie de masse. Dune part, la masse dtecte (+/- intervalle de tolrance) est utilise pour tablir la liste de toutes les combinaisons en acides amins correspondant cet intervalle de masses. Dautre part, les modles tablis pour chacune des sparations sont utiliss en parallle pour trier la liste initiale de combinaisons possibles tablie selon la masse. Ces listes classes sont ensuite confrontes pour aboutir aux combinaisons les plus probables. La stratgie didentification des peptides se fait en deux tapes : lanalyseur Orbitrap apporte une connaissance sur la masse exacte qui est utilise directement dans le logiciel permettant une rduction trs importante de la fentre de masse (de lordre de 20 ppm), lanalyseur LTQ permet la confirmation de la squence en acides amins du peptide considr. Le logiciel a t appliqu des solutions de peptides synthtiques et est en cours dexprimentation pour lidentification de peptides dun mlange complexe rel issu de lhydrolyse enzymatique dune protine vgtale. Link: http://lrgp.ensic.inpl-nancy.fr/index.php?id=286
343/381
[P257] Structural identification by mass spectrometry and antibacterial activity of a peptide from the venom of the Brazilian ant Tetramorium bicarinatum
Aline RIFFLET1, Sabine GAVALDA1, Nathan TENE1, Jrme ORIVEL2, Jrme LEPRINCE3, Laure GUILHAUDIS4, Eric GENIN5, Anglique VETILLARD1, Michel TREILHOU1
1
Equipe d'accueil "Venins et Activits Biologiques" EA4357, CUFR Champollion, place de Verdun, 81012 cedex 09 Albi
2 3
CNRS, UMR Ecologie des Forts de Guyane, Campus Agronomique BP 316, 97379 cedex Kourou
Inserm U982, PRIMACEN Institut Fdratif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Universit de Rouen, 76821 Mont-Saint-Aignan
4
Equipe de Chimie Organique et Biologie Structurale UMR 6014 CNRS, Institut de Recherche en Chimie Organique Fine (IRCOF), IFRMP 23, Universit de Rouen, 76821 Mont-Saint-Aignan
5
ThermoFisher Scientific, 16 avenue du Qubec, 91963 Courtaboeuf
Contact: Michel TREILHOU (michel.treilhou@univ-jfc.fr)
Keywords: Bioinformatics et biostatistics dedicated to proteomics, Signaling, interactomics and post-translational modifications, Systems biology With more than 14 700 species described, ants represent an interesting source for new drugs discovery. Works on a few ant species have yielded many compounds with a variety of novel and potent activities. We studied the venom of a tropical ant, Tetramorium bicarinatum, a terrestrial ant from Brazil. The biological activities and biochemical characterization of the active fraction were investigated on staphylococci. After fractionation of the crude venom by RP-HPLC, MS analysis of an active fraction revealed the presence of two different peptides: the first one, Peptide-1 was represented by the multicharged ions: [M+2H]2+ = 1107.72 and [M+3H]3+ = 738.7. The second peptide was identified with the following ions: [M+H]+ = 1572.1 and [M+2H]2+ = 787.00. These two compounds co-eluted and could not be separated by HPLC. Sequences were determined by de novo sequencing using LTQ-Orbitrap mass spectrometry and confirmed by Edman degradation. Peptide 1 was a 20 amino acid residues and was C-terminally amidated as the majority of peptides from the venom of insects implemented in databases. Peptide 2 was a 15 amino acid residue and was also amidated. Antibacterial activity against Staphylococci aureus and xylosus strains was evaluated using the active HPLC fraction of crude venom then validated with synthetic replicates. Only peptide 1 was active. Interestingly, this peptide had a linear structure, exhibited no meaningful similarity with any itemized peptides from venoms and showed a potent and broad antibacterial activity similar to mellitin, an antimicrobial peptides AMP widely used in microbiological screening. Circular dichrosm showed 44% of helicodal organisation in 8 mM of SDS solution. Among all the new AMPs recently found, this novel small cationic antimicrobial peptide that we have named Bicarinalin, would be a good candidate for development of new antibiotic drugs against bacterial resistant strains. Link: http://www.univ-jfc.fr/equipesrecherche/venins-activites-biologiques-vacbio-ea-4357-albi
344/381
[P258] Nouvelle interface CESI pour l'analyse de dregues

John Hudson1, Michel Anselme1
1
Beckman-Coulter, 33,rue des Vanesses, 95942 Villepinte
Contact: Michel Anselme (manselme@beckman.com)
Keywords: Instrumentation In forensic drug analysis there is always a need for increased sensitivity. In this work we apply a prototype sheathless interface for capillary electrophoresis-ESI-Mass spectrometry (CE-MS)The ruggedness of the HSPS interface was tested in the exploration of the urine metabolic profile after ingestion and metabolism of the antimalarial drug: chloroquine
345/381
[P259] Separation of Fucosylated, non-fucosylated, and complex Carbohydrates By capillary eletrophoresis

Sushma Rampal1, Michel Anselme1
1
Beckman-Coulter, 33 rue des vanesses, 95942 Villepinte
Contact: Michel Anselme (manselme@beckman.com)
Keywords: Separative analysis methods In order to gain in comprehensive understanding of therapeutic MAB, it is necessary to critically caracterize glycosylation associated with them. We develop methodology by which fucosylated,afucosylated,sialylated and complex antennary oligosaccharides can be differentiated from one another
346/381
[P260] Caractrisation de protines entires par couplage indirect CIEF/MALDI-MS

Michael Biacchi1, Nina Pourhassan1, Yannis FRANCOIS1, Emmanuelle Leize-Wagner1
1
Laboratoire de Dynamique et Structure Molculaire par Spectromtrie de Masse, LDSM2, UMR-CNRS 7177, Institut de Chimie, Universit de Strasbourg , 1 rue Blaise Pascal, 67000 Strasbourg
2
Laboratoire Biochimie Toxicologie, CHR Metz Thionville, Hpital Bel Air, 1 Rue du Friscaty, 54100 Thionville
Contact: Yannis FRANCOIS (yfrancois@unistra.fr)
Keywords: Separative analysis methods La caractrisation fine de protines intactes apparat comme un des dfis les plus importants pour les sciences analytiques. En effet, depuis quelques annes, la production et la demande danalyse de protines intactes ont fortement augmentes, notamment dans lindustrie biopharmaceutique. De nos jours, les approches classiques mettant en jeu des couplages chromatographiques avec la spectromtrie de masse (MS) rpondent cette problmatique. Cependant, comme toutes techniques, ces mthodes prsentent certaines limites, notamment en terme de haute masse, dhydrophobicit et de dtermination du point isollectrique (pI). Le couplage de lisolectrofocalisation en mode capillaire (CIEF) avec la MS apparat comme une alternative aux techniques chromatographiques classiques. En effet, la CIEF est une mthode utilisant les proprits de llectrophorse capillaire, dans le but de sparer les peptides et les protines suivant leur pI. Llaboration sous champs dun gradient de pH laide dune solution dampholytes permet dans un premier temps une tape de focalisation des protines suivant leur pI. Une deuxime tape de mobilisation sous champs par lapplication dune faible pression permet leur dtection en MS. Cette mthode couple la MS permet une caractrisation orthogonale en terme de pI et de masse des protines. Cette tude a t ralise sur un couplage CIEF avec une source dionisation laser assiste par matrice (MALDI). En effet, le MALDI a pour avantage dtre plus tolrant avec les conditions de tampon et de sel utiliss en CE, et notamment pour les ampholytes. Par consquent, le couplage indirect CIEF/MALDI-MS avec collecteur de fraction a t mis en place au laboratoire. Les conditions de sparation ont t optimises sur un mlange de protines modles (myoglobine, leucine, alcohol deshydrogenase, albumine bovin).
347/381
[P261] Data independent MALDI ion mobility acquisition for the analysis of tryptic peptides for Proteomic and MALDI Imaging applications
Emmanuelle Claude1, Mark Towers1, Marie-Claude Djidja2, James Langridge1
1 2
Waters Corporation, Atlas Park, Simonsay, M22 5PP Manchester, UK The Institute Cancer of Research, 123 Old Brompton Road, SW7 3RP London, UK
Contact: Emmanuelle Claude (emmanuelle_claude@waters.com)
Keywords: Ionic mobility, Imaging mass spectrometry Introduction Analysis of tryptic peptides by matrix assisted laser desorption/ionisation (MALDI) mass spectrometry, typically involves acquiring an MS experiment first. If identification of the digested protein is not possible by peptide mass fingerprint (PMF), MS/MS analysis of specific peptides would be attempted. This can be time consuming and in the case of MALDI imaging, it may not allow localisation of the tryptic peptides. Identification in imaging experiments is carried out after processing the MS data. Here we are proposing a novel data independent way of acquiring data where MS and MS/MS information are acquired within the same experiment, without any selection of the precursors using ion mobility separation (IMS) to carry out an orthogonal separation of peptides in the gas phase. Methods Data were acquired using the MALDI SYNAPT G2 where tri-wave has separate ions according to their ion mobility in the gas phase. Peptides, from in solution digest, 2-D gel electrophoresis or tissue section are separated both by m/z and ion mobility. Within the same MALDI-IMS experiment, the mass spectrometer is alternated between low collision energy applied to the transfer collision cell and elevated energy, to induce peptide fragmentation. As the fragmentation occurs after the IMS, the precursor at low energy will have the same drift time as its fragments from the elevated energy scan. The complex dataset is subsequently processed where the data from both low and elevated energy is aligned, and correlation between parent and fragment ions is made. Preliminary data The first part of the study will focus upon the analysis of excised spots from 2D- gels, in-gel digested. The extracted solution will be mixed with matrix and spotted onto a MALDI target. Data is processed manually and peak lists (.pkl) are generated for each sample where multiple precursor information is included. PMFs from the low energy are also processed into a text file for protein identification using MASCOT. The MOWSE scores between the two types of analysis are compared. This step will test the speed and robustness of the technique as well as its limitations. The second part of the study will be focus on data acquired directly from tissue section where proteins have been tryptically digested in-situ and analysed by MALDI imaging. Here adjacent pixels had low and elevated CE applied. Ion images of the peptides can be displayed showing their distribution throughout the tissue. The same dataset can then also be processed for identification of the tryptic peptide where regions of specific drift time were selected. Novel aspect Improved methodology for tryptic peptide identification with data independent MALDI analysis using orthogonal ion mobility separation
348/381
[P262] Etude des complexes dactinides en solution : apport de la spectromtrie de masse electrospray
Laurence Berthon1, Nicole Zorz1, Julie Muller1, Geoffroy Ferru1, Cecile Marie2, Manuel Miguirditchian2
1 2
DEN/DRCP/SCPS/Laboratoire des Interactions Ligands Actinides, marcoule, 30200 bagnols sur Cze, France
DEN/DRCP/SCPS/laboratoire d'laboration des procds de sparation, marcoule, 30200 bagnols sur Cze, France Contact: Laurence Berthon (laurence.berthon@cea.fr)
Keywords: Organic and inorganic mass spectrometry Dans le cadre du traitement du combustible nuclaire us, le CEA mne des recherches sur la sparation des actinides par extraction liquide-liquide. Cette opration est un phnomne complexe dont les mcanismes dinteraction des diffrents constituants du systme biphasique (extractants, diluants, complexants, soluts) ne sont pas encore parfaitement dcrits. Pour amliorer la connaissance des systmes chimiques mis en uvre, des tudes de spciation molculaire sont ncessaires aussi bien en phase aqueuse quen phase organique. Ces tudes de spciation visent mieux dcrire les quilibres d'extraction mais galement amliorer la comprhension de laffinit et la slectivit des molcules extractantes. Pour cela, les complexes forms entre les soluts et les ligands (extractant ou complexant) sont caractriss par diffrents techniques complmentaires. La spectromtrie de masse ionisation lectropray est une technique de choix permettant dobtenir des informations sur la structure (stchiomtrie) et la stabilit des complexes ainsi que sur la slectivit des molcules extractantes. Pour tudier des solutions radioactives contenant des actinides, un spectromtre de masse a t adapt et connect une boite gants dans linstallation Atalante du CEA Marcoule. Les rsultats prsents illustrent la caractrisation par spectromtrie de masse electrospray des complexes forms entre les actinides tels que luranium (VI), lamricium (III)ou le plutonium (IV) et plusieurs ligands dintrts.
349/381
[P263] A new strategy for studying structural transitions in proteins developed by Tyrosine-targeted spin labeling and EPR spectroscopy
Sabrina Lignon1, Rgine Lebrun1, Brigitte Meunier-Gontero1, Magali Lorenzi2, Carine Puppo2, Marlne Martinho2, Elisa Mileo3, Sylvain Marque3, Bruno Guigliarelli2, Valrie Belle2
1
Plate-forme Protomique de l'Institut de Microbiologie de la Mditerrane, CNRS-IFR88, Marseille Protomique, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France
2
Bionergtique et Ingnierie des Protines UPR 9036, CNRS & Aix-Marseille Universit, Institut de Microbiologie de la Mditerrane, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France
3
Laboratoire Chimie Provence, LCP-UMR 6264, Universit de Provence, case 521, Avenue Escadrille Normandie-Niemen, 13397 Marseille cedex 20, France Contact: Sabrina Lignon (lignon@ifr88.cnrs-mrs.fr)
Keywords: Signaling, interactomics and post-translational modifications, Organic and inorganic mass spectrometry Structural transitions in proteins, and especially in the flexible and disordered ones, are studied by Site Directed Spin Labeling EPR spectroscopy which conventionally uses nitroxide probes functionalized to target cysteine residues (1). Cysteine residues are rare amino acids in proteins and they have frequently functional roles. They are involved in structural elements such as disulfide bridges or in the binding of metal cofactors, therefore grafting of the nitroxide probe to residues other than cysteines is required. We describe here the first results of tyrosinetargeted spin labeling on a chloroplast protein of 12 kDa, so-called CP12, having a unique tyrosine and 4 cysteine residues involved in two functional disulfide bridges (2). A threecomponent Mannich-type reaction was performed leading to a labeling of 70(10)% of the protein. Electrospray and MALDI-ToF mass spectrometry analyses showed that the tyrosine residue was labeled. The global structure of the modified CP12 was not affected by the chemical modification as demonstrated by CD spectroscopy. The interaction with its physiological partner GAPDH, as previously described (3), could also be achieved by in vitro reconstitution assay. EPR spectroscopy of the labeled protein showed a very high mobility of the probe that still remained very mobile after complex formation with GAPDH. In this work, we demonstrate for the first time that a nitroxide probe can be selectively grafted on tyrosine residues by using the Mannich-type reaction. This demonstration opens the way to the study of proteins carrying functional cysteine residues by spin labeling EPR spectroscopy. 1-Erales J, Lorenzi M, Lebrun R, Fournel A, Etienne E, Courcelle C, Guigliarelli B, Gontero B, et Belle V. A new function of GAPDH from Chlamydomonas reinhardtii : A thiol/disulfide exchange reaction with CP12. 2009, Biochemistry, vol. 48, pp. 6034-6040. 2- Lorenzi M, Puppo C, Lebrun R, Lignon S, Roubaud V, Martinho M, Mileo E, Tordo P,. Marque R.A S, Gontero B, Guigliarelli B et Belle V. Tyrosine-targeted spin labeling and EPR Spectroscopy: an alternative strategy for studying structural transitions in proteins. 2011, accepted in Angewandte Chemie. 3-Erales J, Lignon S, et Gontero B. CP12 from Chlamydomonas reinhardtii : a permanent specific "chaperone-like" protein of glyceraldehyde-3-phosphate dehydrogenase. 2009, J. Biol. Chem., vol. 284, pp. 12735-12744.
350/381
[P264] Liquid Extraction Surface Analysis (LESA)of proteins on tissue sections.

Jusal Quanico1, Julien Franck1, Maryna Kuzminska1, Michel Salzet1, Isabelle Fournier1, Maxence Wisztorski1
1
Laboratoire de Spectromtrie de Masse Biologique, Fondamentale & Applique, FABMS, USTL, Universit Lille Nord de France Universit Lille 1 Bt SN3, 1er tage, 59655 Villeneuve d'ascq, France Contact: Jusal Quanico (jusal.quanico@etudiant.univ-lille1.fr)
Keywords: Imaging mass spectrometry, High mass and high resolution mass Spectrometry, Separative analysis methods A method for the mass spectrometry (MS) analysis of proteins after in situ enzymatic digestiongenerated peptides was developed employing liquid extraction surface analysis (LESA) using the Advion TriVersa NanoMate platform. This ambient surface sampling technique extracts analytes by making a single droplet of solvent in contact with the sample surface, creating a liquid microjunction where extraction of the analyte occurs. The extract is then aspirated and introduced to the ion source. Because the platform is an auxiliary device, it can be onlinecoupled with a variety of atmospheric pressure ion sources. We also developed the possibility to use it offline for analyses by using vacuum ion source like MALDI. The capability of LESA to analyze exogenous small molecules and lipids has been demonstrated. In this work, the LESA technique was used offline, enabling the extraction of protein digests from frozen and formalinfixed, paraffin embedded (FFPE) rat brain tissue sections after trypsin digestion. LESA allowed the in situ extraction of localized digested peptides and thus enabled the profiling of proteins in the tissue sections analyzed. Although it offers a lesser spatial resolution compared with MSI techniques like matrix-assisted laser desorption/ionization (MALDI) imaging, LESA allowed for the collection of extracts and their further treatment prior to introduction to the mass spectrometer. As an example, the extracted peptides were subjected to solid phase extraction for salt removal and introduced to a nano-high performance liquid chromatography (nanoHPLC) for further separation. LESA can thus serve as a complementary MS ambient surface sampling technique that can be used to provide further information needed in peptide analysis.
351/381
[P265] Genoproteomilks: A strategy combining genomic and proteomic tools to qualify the protein fraction of milks
Guy Miranda1, Leonardo Bianchi1, Amlie Pinard1, Cline Henry2, Alain Guillot2, Tania Klein3, Patrice Martin1
1
INRA, UMR1313 Unit Gntique Animale et Biologie Intgrative, quipe Lait, Gnome & Sant , Domaine de Vilvert, Btiment 221, 78350 Jouy-en-Josas, France
2
Plateforme dAnalyse Protomique Paris Sud-Ouest (PAPPSO), Domaine de vilvert, 78350 Jouy-en-Josas, France
3
UPRA Tarentaise , 40 rue du Terraillet , 73190 Saint Baldoph, France
Contact: Patrice Martin (patrice.martin@jouy.inra.fr)
Keywords: Systems biology, Separative analysis methods, Proteins, peptides and small molecules quantification Over the past 40 years, dairy cattle have been selected mainly on milk and total protein yield. Selection was carried out at the expense of overall milk quality, as well as health and fertility of high-producing dairy cows. Moreover, the process of genetic selection itself leads to a loss of genetic variability that is particularly severe in highly specialized breeds. Awaiting the application of genomic selection on a large scale, some functional traits, such as fertility, resistance to pathogens, longevity and milk quality have been implemented to breeding programs to warrant acceptable fertility, welfare of dairy cows and sustained milk production, opening new valorization fields. For many years, milk has been considered as a raw material of which processing or cracking concentrated most of the added value. More recently, emphasis has been put on milk elementary components since many of them, in particular fatty acids and peptides, have putative or actual positive effects on human health. There is nowadays a growing interest to accurately measure elementary milk components, including proteins, at low cost, to identify genetic, and to a less extent environmental factors, affecting milk protein gene expression. This would make it possible to design effective tools to obtain milk with the desired composition. Genetic variants of milk proteins have been shown to impact milk composition both at the qualitative and quantitative levels, and determine nutritional as well as technological properties of milks. It is therefore crucial to be able to characterize precisely milk protein composition. Several techniques have been used to estimate the variation in concentration, posttranslational modification (phosphorylation and glycosylation) and genetic polymorphism of milk proteins, including liquid chromatography and capillary electrophoresis. Here we present a novel and powerful approach combining LC-MS and exon re-sequencing that proved to be particularly effective to provide a detailed description of the milk protein fraction in the French Tarine cattle breed in which a new beta-casein genetic variant has been identified.
352/381
[P266] Mechanistic aspect of the maleic anhydride grafting onto fatty double bonds: Evidence of radical addition reactions by mass spectrometry
O. Zovi1, C. Loutelier-Bourhis2, L. Lecamp1, C. Bunel1, C. Lange1
1
INSA de Rouen UMR CNRS 6270 PBS FR CNRS 3038, avenue de lUniversit, 76 801 Saint-Etienne-du-Rouvray Cedex
2
Universit de Rouen UMR CNRS 6014 COBRA FR CNRS 3038 , rue Tesnires , 76 821 Mont- Saint-Aignan Cedex Contact: C. Loutelier-Bourhis (CORINNE.LOUTELIER@univ-rouen.fr)
Keywords: Systems biology The grafting of maleic anhydride onto fatty double bond is a usefull method to functionalize polymers or biomolecules such as triglycerides molecules [1,2]. However, although some mechanisms involved in grafting reaction have been proposed [3,4], the structures of the grafted products have not been actually well defined. In this study, the thermal grafting of maleic anhydride onto (un)saturated fatty esters without use of initiator was characterized and the structure of the products determined. The resort to several mass spectrometric techniques including GC/MS, LC/MSn and accurate mass measurements permitted to establish that various addition mechanisms occured during the grafting reaction; (i) radical addition either onto the double bond or at the allylic position followed by combination or elimination reaction and (ii) Diels Alder addition between diunsaturated fatty acyl chains and maleic anhydride could be evidenced. [1] Feldman D. J. Macromol. Sci. Part A: Pure Appl. Chem. 2005; 42: 587-605. [2] Ciardelli F., Coiai S., Passaglia E., Pucci A., Ruggeri G. Polym. Int. 2008; 57: 805-836. [3] Passaglia E., Coiai S., Augier. S. Progress Polym. Sci. 2009; 34: 911-947. [4] Nakason C., Kaesaman A., Supasanthitikul P. Polym. Testing. 2004; 23: 35-41.
353/381
[P267] HPTLC-MS, couplage dune mthode de chromatographie liquide haut dbit avec la spectromtrie de masse. Que choisir aujourdhui ?
Gerda Morlock1, Pierre Bernard-Savary2, Aziz Filali-Ansary3
1 2 3
Universit de Hohenheim, , 70599 Stuttgart, Allemagne Chromacim SAS, L'ancienne Eglise, 38340 Pommiers-la Placette, France DSAR Sanofi-Aventis 1,Avenue Pierre Brossolette 91385 Chilly-Mazarin, France
Contact: Pierre Bernard-Savary (pbs@chromacim.com)
Keywords: Organic and inorganic mass spectrometry, Instrumentation, Separative analysis methods Le congrs dHPTLC, qui a eu lieu Ble dbut Juillet a t un norme succs avec un nombre de participants qui a doubl depuis la dernire dition en 2008. Ceci est en partie d aux rcents dveloppements en matire de couplage de cette mthode avec la spectromtrie de masse. Cette contribution crite se propose de prsenter ltat de lart et ses perspectives court terme envisages sous langle du couplage la spectromtrie de masse et du haut dbit. LHPTLC est particulirement adapte au haut dbit du fait de sa rapidit, comparativement lUPLC qui fait actuellement rfrence. Quelques exemples comparatifs sur des mthodes actuelles viennent confirmer cette situation par des chiffres. Ceci confirme lide selon laquelle un quipement dHPTLC ralise approximativement le travail de trois quipements dHPLC, ce qui sexplique par le fait que la sparation est faite en parallle, et par le fait quaucune tape de rinage nest ncessaire entre les chantillons, quelle que soit la matrice associe. Dans de nombreux cas certaines tapes de prparation dchantillon peuvent tre simplifies, voire carrment supprimes. Cette contribution dcrit galement les diffrentes solutions pour le couplage avec la spectromtrie de masse. Elle diffrencie deux catgories dinterfaces selon quelles fonctionnent par lution ou bien par dsorption du compos. Des tudes comparatives ont t ralises et les limites de dtection sont du niveau de ce que lon peut observer en densitomtrie dans le spectre UV dabsorption ou de fluorescence. Cependant, une bonne reproductibilit ncessite un minimum de prcautions. Mais finalement le couplage avec lHPTLC reste simple et efficace. Grce aux nouvelles interfaces HPTLC-MS, un certain nombre de laboratoires risquent donc fort de sintresser lHPTLC, considre comme une nouvelle mthode. R. Cody, J. Laramee, H. Dupont Durst, Anal Chem 77 (2005) 2297-2302 U. Jautz, G. Morlock, J Chromatogr A 1128 (2006) 244-250 H. Luftmann, M. Aranda, G. Morlock, Rapid Commun Mass Spectrom 21 (2007) 37723776 Link: www.hptlc.com
354/381
[P268] DBS-MS 500, automate pour lchantillonnage haut dbit pour 500 cartes la fois. Fiabilit, rapidit et sensibilit des premiers rsultats
Pierre Bernard-Savary1
1
Chromacim SAS, L'ancienne Eglise, 38340 Pommiers-la Placette, France
Contact: Pierre Bernard-Savary (pbs@chromacim.com)
Keywords: Instrumentation, Proteins, peptides and small molecules quantification , Systems biology La mthode danalyse des constituants du sang base sur une goutte de sang sch sur papier filtre, appele du sigle DBS pour Dried Blood Spot, est en plein essor actuellement dans diffrents secteurs, et principalement pour les essais cliniques.. Fort dune exprience de plus de 50 ans dans les appareils automatiques de manipulation dchantillons liquides et danalyse, la socit Suisse Camag sest lance dans ce domaine dans le cadre de lexploitation dun ingnieux brevet. Ce brevet dextraction en phase liquide dans un substrat en couche mince tait initialement prvu pour un usage en Chromatographie sur Couche Mince (CCM) ou HPTLC (high-performance thin-layer chromatography) pour sa version performante. Le congrs de cette spcialit, pour laquelle Camag justifie sa rputation de leader, sest droul dbut Juillet Ble en Suisse [1]. Cette contribution crite se propose de prsenter les solutions technologiques dveloppes par la socit Camag ainsi que les premiers rsultats trs encourageants des premiers tests effectus de ce robot qui sera commercialis en fin danne 2011. La mthode propose prsente de nombreux avantages. Les principaux sont une conomie de temps et de cot trs importants en comparaison de la mthode de rfrence par HPLC ou UPLC MS. Ceci est du au transfert direct de lanalyte de la bande de papier vers le spectromtre de masse. Un autre avantage est le gain de sensibilit qui est suprieur 10x, galement du fait de lextraction directe. [1] www.hptlc.com Link: http://www.camag.com/v/products/dbs-extraction-device/video.html
355/381
356/381
Participants list
357/381
This list is based on registration on August 23 th. A list of participants which registered after this date will be available at the office desk (Salle des gardes room (n1 on plan page 12))
-AMiss Christelle ABSALON - INSTITUT DES SCIENCES MOLECULAIRES : c.absalon@ism.u-bordeaux1.fr Miss Claire ADAM - CEA : claire.adam@cea.fr Mrs Annie ADRAIT - IRTSV/BGE/EDYP U1038 CEA-GRENOBLE : annie.adrait@cea.fr Dr Carlos AFONSO - UPMC/IPCM : carlos.afonso@upmc.fr Miss Rima AIT-BELKACEM - PIT2 : a_b_rima@hotmail.com Mr Ahmad AL ALI - INSERM DRPA05 : alali@icsn.cnrs-gif.fr Mr Renaud ALBIGOT - IPBS-CNRS : renaud.albigot@ipbs.fr Dr Florian ALBRIEUX - UNIVERSITE CLAUDE BERNARD LYON 1 : florian.albrieux@univ-lyon1.fr Mrs Pascale ALIPRANDI - METABOLIC EXPLORER : paliprandi@metabolic-explorer.com Mr Franois ALLAIN - DSV/IBEB/SBTN : francois.allain@cea.fr Dr Lionel ALMERAS - IRBA ANTENNE MARSEILLE : almerasl@imtssa.fr Mrs Christine ALMUNIA - DSV/IBEB/SBTN : christine.almunia@cea.fr Mrs Valrie ALOIN - DSV/IBEB/SBTN : valerie.aloin@cea.fr Mrs Batrice ALONSO - DSV/IBEB/SBTN : beatrice.alonso@cea.Fr Mrs Batrice ALPHA - BAZIN - DSV/IBEB/SBTN : beatrice.alpha-bazin@cea.fr Dr Magali ANDRE - LFB BIOTECHNOLOGIES : ribeiro@lfb.fr Mrs Patricia ANGLADE - MICALIS : patricia.anglade@jouy.inra.fr Dr Karim ARAFAH - PLATEFORME BIOPARK D'ARCHAMPS : karim.arafah@biopark-archamps.org Miss Marie ARLOTTO - INSERM U1038 / EDYP : marie.arlotto@cea.fr Mr Jean ARMENGAUD - DSV/IBEB/SBTN : jean.armengaud@cea.fr Miss Carine ARNAUDGUILHEM - IPREM UMR 5254 : carine.arnaudguilhem@univ-pau.fr Mr Lo AUBERT - SERVICE FACTURIER : l.aubert62@gmail.com Dr Frdric AUBRIET - LSMCL - UNIVERSIT PAUL VERLAINE : frederic.aubriet@univ-metz.fr Dr Stephane AUDEBERT - CRCM : stephane.audebert@inserm.fr Mr Rmy AURAND - INRA PACA : remy.aurand@avignon.inra.fr Miss Aline AURIAS - SPECTRA ANALYSE - EDITIONS PCI : a.aurias@editions-pci.fr Miss Sophie AYCIRIEX - PLATEFORME FINANCIERE PHARMACIE : sophie.ayciriex@parisdescartes.fr Mrs Christine AYOUB - PROTEABIO EUROPE : c.ayoub@eu.proteabio.com
-B-
Mr Sacha BAGINSKY - MARTIN LUTHER UNIVERSITT HALLE WITTENBERG : sacha.baginsky@biochemtech.uni Mrs Catherine BALTHASAR - CLUZEAU INFO LABO : cil@cluzeau.com Mrs Sylvie BALZANI - SANOFI-AVENTIS : sylvie.balzani@sanofi-aventis.com Ms Perdita BARRAN - UNIVERSITY OF EDINBURGH : perdita.barran@ed.ac.uk Miss Caroline BARRRE - INSA DE ROUEN : caroline.barrere@hotmail.fr Mr Damien BARTHE - CEA/GRENOBLE : damien.barthe@cea.fr Dr Isabelle BATXELLI-MOLINA - CNRS : isabelle.molina@sysdiag.cnrs.fr Miss Emilie BAUDELET - INSERM DRMRS : emilie.baudelet@inserm.fr Mr Mathieu BAUDET - CEA GRENOBLE : mathieu.baudet@cea.fr Prof Bruno BAUDIN - UFR PHARMACIE : bruno.baudin@sat.aphp.fr Mrs Karima BAUDIN - PERKINELMER : Karima.Baudin@perkinelmer.com Miss Mathilde BEAU - INGNIEUR D'TUDE : mathilde.beau@ipbs.fr Dr Martine BEAUFOUR - CNRS-CBM : martine.beaufour@cnrs-orleans.fr Miss Chrine BECHARA - UPMC - LBM : cherine.bechara@upmc.fr Mr Francois BECHER - CEA SACLAY : francois.becher@cea.fr Miss Maya BELGHAZI - CNRS : maya.belghazi@univmed.fr Mr Patrick BELLEMIN - BIO RAD : patrick.bellemin@bio-rad.com Dr Hlne BELVA-BESNET - SANOFI-AVENTIS : helene.belva-besnet@sanofi-aventis.com Mr Farid BENKACI-ALI - CHERCHEUR : benkaciaf@yahoo.fr Mr Yves BRARD - SANOFI PASTEUR : yves.berard@sanofipasteur.com Miss Carole BERAUD - ETUDIANTE : carole.beraud@etu.utc.fr Mr Pierre BERNARD SAVARY - CHROMACIM SAS : pbs@chromacim.com
358/381
Dr Stphane BERNILLON UMR1332 BIOLOGIE DU FRUIT ET PATHOLOGIE stephane.bernillon@bordeaux.inra.fr Dr Laurence BERTHON - CEA MARCOULE : laurence.berthon@cea.fr Mr Fabrice BERTILE - CNRS IPHC DSA : fbertile@unistra.fr Miss Gwladys BERTIN - IRD-UMR216 : gwladys.bertin@gmail.com Mrs Sophie BERTIN - INSTITUT DE RECHERCHES SERVIER : sophie.bertin@fr.netgrs.com Mr Michael BIACCHI - INSTITUT DE CHIMIE DE STRASBOURG : npotier@chimie.u-strasbg.fr Dr Jordane BIARC - UCSF : jbiarc@cgl.ucsf.edu Miss Claudia BICH - ICSN CNRS UPR 2301 : claudia.bich@icsn.cnrs-gif.fr Dr Willy BIENVENUT - CHERCHEUR : willy.bienvenut@isv.cnrs-gif.fr Mrs Cline BLAND - DSV/IBEB/SBTN : celine.bland@cea.fr Dr Mlisande BLEIN-NICOLAS - INRA : melisande.blein@moulon.inra.fr Prof Joel BOCKAERT - CNRS IGF : joel.bockaert@igf.cnrs.fr Miss Audrey BODIN - DOCTORANTE : audrey.bodin@cemes.fr Dr Elisabetta BOERI ERBA - ETH : eboerierba@gmail.com Mr Philippe BOGARD - SERVA ELECTROPHORESIS GMBH : philippe.bogard@proteomics.consul.com Dr Wilfrid BOIREAU - INSTITUT FEMTO-ST : wboireau@femto-st.fr Dr Grard BOLBACH - UMPC - LBM : gerard.bolbach@upmc.fr Prof Marc BONNEU - UNIVERSITE BORDEAUX SEGALEN POLE PROTOMIQUE : marc.bonneu@ipb.fr Dr Andy BORTHWICK - NONLINEAR DYNAMICS : andy.borthwick@nonlinear.com Dr Ali BOUAMRANI - INSERM U836-EQ7 : abouamrani@yahoo.fr Mr Stphane BOUCHONNET - ECOLE POLYTECHNIQUE : stephane.bouchonnet@dcmr.polytechnique.fr Mr Guy BOUCHOUX - ECOLE POLYTECHNIQUE DCMR : bouchoux@dcmr.polytechnique.fr Dr Pierre Edouard BOUGIS - PIERRE EDOUARD BOUGIS : pierre-edouard.bougis@univmed.fr Mrs Stphanie BOULLANGER - CERMAV/CNRS : stephanie.boullanger@cermav.cnrs.fr Mr Valentin BOUQUET - ASA - ADVANCED SOLUTIONS ACCELERATOR : vbouquet@asa-sas.com Miss Valrie BOURDON - UNIV. PAUL SABATIER : bourdon@chimie.ups-tlse.fr Mr Emmanuel BOURGOGNE - UNIVERSIT PARIS DESCARTES : emmanuel.bourgogne@parisdescartes.fr Miss Cline BOURSIER - UNIVERSIT PARIS SUD 11 : celine.boursier@u-psud.fr Dr Marie-pierre BOUSQUET-DUBOUCH - IPBS - CNRS : marie-pierre.bousquet@ipbs.fr Mr David BOUYSSI - IPBS/CNRS : david.bouyssie@ipbs.fr Mr Brice BOUYSSIERE - IPREM UMR 5254 : brice.bouyssiere@univ-pau.fr Mr Bessem BRAHIM - IPMC/CSOB : bessem.brahim@upmc.fr Mrs Catherine BRETHENOUX - SANOFI : catherine.brethenoux@sanofi-aventis.com Ms Jennifer BRODBELT - UNIVERSITY OF TEXAS : jbrodbelt@mail.utexas.edu Mr Cedric BROUSSARD - INSERM : cedric.broussard@inserm.fr Mrs Sabine BRUGIRE - INGNIEUR : sabine.brugiere@cea.fr Mr Christophe BRULEY - CEA GRENOBLE : christophe.bruley@cea.fr Mrs Virginie BRUN - UNITE DE BIOLOGIE A GRANDE ECHELLE : virginie.brun@cea.fr Miss Claire BRUNET - UNIVERSIT CLAUDE BERNARD LYON 1 : cbrunet@lasim.univ-lyon1.fr Mr William BUCHMANN - UNIVERSIT D'EVRY VAL D'ESSONNE : william.buchmann@univ-evry.fr Dr Philippe BULET - LABORATOIRE AGIM : philippe.bulet@agim.eu Miss Audrey BULETE - CNRS UMR 5280 : a.bulete@sca.cnrs.fr Mrs Corinne BUR - CBMN UMR 5248 / CGF : c.bure@cbmn.u-bordeaux.fr Mr Alexandre BUREL - UNIVERSITE DE STRASBOURG : alexandre.burel@unistra.fr Miss Ccile BUSSET - AB SCIEX : cecile.busset@absciex.com
-CDr Martine CADENE - CBM CNRS : cadene@cnrs-orleans.fr Dr David CALLIGARIS - CRO2-PIT2 : callidav@yahoo.fr Dr Luc CAMOIN - MARSEILLE PROTOMIQUE - CRCM : luc.camoin@inserm.fr Dr Francis CANON - POST-DOCTORANT : francis.canon@synchrotron-soleil.fr Dr Sonia CANTEL - IBMM UMR 5247 UNIVERSIT MONTPELLIER 1 ET 2 : sonia.cantel@univ-montp2.fr Miss Christine CARAPITO - CNRS IPHC DSA : ccarapito@unistra.fr Mr Vincent CARR - LSMCL - UNIVERSIT PAUL VERLAINE : carre@univ-metz.fr Mrs Patricia CASTEL - INSTITUT DES SCIENCES MOLECULAIRES : p.castel@u-bordeaux1.fr Mr Guillaume CAZALS - UNIVERSIT DE MONTPELLIER 2 : guillaume.cazals@univ-montp2.fr
359/381
Mrs Delphine CENTENO - CENTRE INRA CLERMONT-FD-THEIX : delphine.centeno@clermont.inra.fr Mr Christopher CERRUTI - ICSN CNRS UPR 2301 : christopher.cerruti@icsn.cnrs-gif.fr Mr Philippe CHAFEY - INSERM DRPA05 : philippe.chafey@inserm.fr Mr Ludovic CHAILLET - SAIC : ludovic.chaillet@gmail.com Mr Patrick CHAIMBAULT - LSMCL - UNIVERSIT PAUL VERLAINE : chaimbault@univ-metz.fr Mr Christophe CHAMBON - ASSISTANT INGNIEUR : cchambon@clermont.inra.fr Dr Julia CHAMOT-ROOKE - LABORATOIRE DCMR : julia.chamot-rooke@polytechnique.edu Mr Thibault CHAMPION - BIOMRIEUX : thibault.champion@biomerieux.com Miss Karima CHAOUI - IPBS/CNRS : Karima.Chaoui@ipbs.fr Mrs Solenne CHARDONNET - IBBMC, UNIVERSIT PARIS-SUD : solenne.chardonnet@u-psud.fr Prof Laurence CHARLES - UNIVERSIT DE PROVENCE : laurence.charles@univ-provence.fr Mrs Marie-Hlne CHARLES - BIOMRIEUX : marie-helene.charles@biomerieux.com Mr Guillaume CHARLOT - LGS LAB GAZ SYSTEMS : guillaume.charlot@labgaz.fr Mr Yannick CHARRETIER - BIOMRIEUX : yannick.charretier@biomerieux.com Mrs Claude CHARVY - CNRS : claude.charvy.clericeau@orange.fr Mr Pierre CHAURAND - : pierre.chaurand@umontreal.ca Mrs Guylaine CHAUSSINAND - DSV/IBEB/SBTN : guylaine.chaussinand@cea.fr Mrs Virginie CHAVANT - IGBMC : chavant@igbmc.fr Dr Jrme CHENAU - CEA DE SACLAY : jerome.chenau@cea.fr Mr Christophe CHENDO - FDRATION DES SCIENCES CHIMIQUES DE MARSEILLE : christophe.chendo@univcezanne.fr Mr Sylvain CHREAU - ONIRIS - LABERCA : sylvain.chereau@oniris-nantes.fr Miss Sylvie CHEVOLLEAU - AXIOM - INRA : sylvie.chevolleau@toulouse.inra.fr Miss Cerina CHHUON - PLATEAU PROTOMES NECKER, PARIS : cerina.chhuon@gmail.com Mr Giovanni CHIAPPETTA - ESPCI PARISTECH : giovanni.chiappetta@espci.fr Dr Fabien CHIROT - EZUS/ UNIVERSIT CLAUDE BERNARD LYON 1 : fabien.chirot@univ-lyon1.fr Mr Joseph CHRISTIE - OLEZA - DSV/IBEB/SBTN : joseph.christie@cea.fr Mr Grmy CLAIR - DSV/IBEB/SBTN : geremy.clair@cea.fr Dr Catherine CLAPAROLS - UNIV. PAUL SABATIER : claparol@chimie.ups-tlse.fr Mr Christophe CLARYSSE - PERKINELMER : christophe.clarysse@perkinelmer.com Dr Stphane CLAVEROL - UNIVERSITE BORDEAUX SEGALEN POLE PROTOMIQUE : stephane.claverol@ubordeaux2.fr Dr Philippe CLEON - SANOFI AVENTIS : Philippe.Cleon@sanofi-aventis.com Dr Emmanuelle COM - SAIC : emmanuelle.com@univ-rennes1.fr Miss Florence COMBES - CEA GRENOBLE : florence.combes@cea.fr Mr Graham COOKS - PURDUE UNIVERSITY : cooks@purdue.edu Dr Laurent COQUET - CNRS UMR6270 : laurent.coquet@univ-rouen.fr Mr Cyril CORBET - SERVICE FACTURIER : cyril.corbet@ed.univ-lille1.fr Mrs Agns CORBIN - NONLINEAR DYNAMICS : agnes.corbin@nonlinear.com Mr David CORNU - CNRS : cornu@icsn.cnrs-gif.fr Mrs Aurlie CORNUT-THIBAUT - INSERM DRLYS FACTURATION : aurelie.thibaut@ens-lyon.fr Mr Pascal COSETTE - UNIVERSITE DE ROUEN : pascal.cosette@univ-rouen.fr Miss Mlaine COURCOUL - INSERM DRPA05 : melaine.courcoul@parisdescartes.fr Mr Yohann COUT - LABORATOIRE BIOLOGIE GRANDE ECHELLE : yohann.coute@cea.fr Mrs Morgane COUVET - INSTITUT ALBERT BONNIOT : MCouvet@chu-grenoble.fr Dr Ccile CREN - CNRS UMR 5280 : c.cren@sca.cnrs.fr Mr Emmanuel CRENN - PROMEGA : emmanuel.crenn@promega.com Dr Myriam CUBIZOLLES - CEA : myriam.cubizolles@cea.fr
-DMr Christophe DABADIE - THERMO ELECTRON SAS : christophe.dabadie@thermofisher.com Mrs Anne-sophie DABERT-GAY - IPMC - CNRS : dabert-gay@ipmc.cnrs.fr Mrs Sada DANOUN - UMR 5546 UPS-CNRS : danoun@lrsv.ups-tlse.fr Mrs Carine DAROLLES - DSV/IBEB/SBTN : carine.darolles@cea.fr Mrs Claire DAULY - THERMO ELECTRON SAS Mrs Anne - Hlne DAVIN - DSV/IBEB/SBTN : anne-hElEne.davin@cea.fr Mrs Christel DAYANG - STALLERGENES : cdayang@stallergenes.fr
360/381
Mr Laurent DEBRAUWER - AXIOM - INRA : laurent.debrauwer@toulouse.inra.fr Mr Yoann DECEUNINCK - ONIRIS - LABERCA : yoann.deceuninck@oniris-nantes.fr Mrs Clia DECHAVANNE - IRD UMR 216 : celia.dechavanne@ird.fr Mrs Paulette DECOTTIGNIES - CNRS/ UNIVERSIT PARIS SUD : paulette.decottignies@u-psud.fr Miss Mathilde DECOURCELLE - INRA : decource@supagro.inra.fr Mr Alain DEDIEU - DSV/IBEB/SBTN : alain.dedieu@cea.fr Mr Pieter DEKOCKER - MS NOISE : contact@msnoise.com Mr Julien DELOBEL - SERVICE RGIONAL VAUDOIS DE TRANSFUSION SANGUINE : julien.delobel@mavietonsang.ch Mr Frdric DELOLME - INSERM DRLYS FACTURATION : frederic.delolme@ibcp.fr Mrs Edith DEMETTRE-VERCEIL - FUNCTIONAL PROTEOMICS PLATFORM - FPP : edith.demettre@igf.cnrs.fr Dr Nicolas DESBENOIT - INSTITUT DE LA VISION, INSERM S968 : nicolas_desbenoit@yahoo.fr Mr Louis DESCHANEL - THERMO FISHER SCIENTIFIC : louis.deschanel@thermofisher.com Mr Michel DESJARDINS - UNIVERSITE DE MONTREAL : michel.desjardins@umontreal.ca Miss Cindy DIERYCKX - AI CNRS : cindy.dieryckx@bayer.com Mr Laurent DIOLOGENT - UNIVERSIT DE LILLE 1 : Petrak@hotmail.fr Mr Bruno DOMON - LUXEMBOURG CLINICAL PROTEOMICS CENTER : bdomon@crp-sante.lu Mr Christian DUCHAMP - UNIVERSITE CLAUDE BERNARD LYON 1 : christian.duchamp@univ-lyon1.fr Dr Magalie DUCHATEAU - PLATE-FORME DE PROTOMIQUE - INSTITUT PASTEUR : magalie.duchateau@pasteur.fr Dr Patrick DUCOROY - CLIPP (CLINICAL AND INNOVATION PROTEOMIC PLATFORM) : Patrick.DUCOROY@clipproteomic.fr Dr Manuelle DUCOUX-PETIT - IPBS/CNRS/UPS : manuelle.ducoux@ipbs.fr Miss Cline DUCRUIX - BIOMRIEUX : celine.ducruix@cea.fr Dr Philippe DUGOURD - CNRS ET UNIVERSIT DE LYON : dugourd@lasim.univ-lyon1.fr Miss Vronique DUPIERRIS - CEA - DSV -IRTSV- BGE -EDYP : veronique.dupierris@cea.fr Mr Mathieu DUPR - UNIVERSIT MONTPELLIER 2 : mathieu.dupre@univ-montp2.fr Mr Jean- William DUPUY - UNIVERSITE BORDEAUX SEGALEN POLE PROTOMIQUE : jean-william.dupuy@ubordeaux2.fr Mr Jocelyn DUPUY - THERMO ELECTRON SAS : jocelyn.dupuy@thermo.com Dr Sylvre DURAND - INSTITUT CURIE - HPITAL REN HUGUENIN : sylvere.durand@curie.net Dr Elodie DURIEZ - CRP-SANT : elodie.duriez@crp-sante.lu Mrs Emie DURIGHELLO - DSV/IBEB/SBTN : emie.durighello@cea.fr
-E-
Dr Aleksander EDELMAN - CENTRE DE RECHERCHE INSERM U845 : cerina.chhuon@gmail.com Mr Marven EL OSTA - CLIPP (CLINICAL AND INNOVATION PROTEOMIC PLATFORM) : marven.elosta@clipproteomic.fr Mr Nicolas ELIE - ICSN CNRS UPR2301 : nicolas.elie@icsn.cnrs-gif.fr Prof Christine ENJALBAL - UNIVERSIT DE MONTPELLIER 2 : christine.enjalbal-goubet@univ-montp2.fr Mr Quentin ENJALBERT - EZUS/ UNIVERSIT CLAUDE BERNARD LYON 1 : quentin.enjalbert@lasim.univlyon1.fr Mr Eric EZAN - DSV/IBEB/SBTN : eric.ezan@cea.fr
-F-
Mr Bertrand FABRE - DOCTORANT : fabre@ipbs.fr Mr Franois FENAILLE - CEA SACLAY : francois.fenaille@cea.fr Mr Bernard FERNANDEZ - DSV/IBEB/SBTN : bernard.fernandez@cea.fr Miss Myriam FERRO - CEA/GRENOBLE : myriam.ferro@cea.fr Mr Stphane FESSEMAZ - BIO RAD : stephane.fessemaz@bio-rad.com Dr Christophe FLAHAUT - LABORATOIRE DE PHYSIOPATHOLOGIE DE LA BHE : christophe.flahaut@univartois.fr Mrs Cline FLEURY - CEA-GRENOBLE : celine.fleury@cea.fr Mrs Catherine FONBONNE - EZUS/ UNIVERSIT CLAUDE BERNARD LYON 1 : catherine.fonbonne@cpe.fr Dr Eric FOREST - INSTITUT DE BIOLOGIE STRUCTURALE : eric.forest@free.fr Dr Laetitia FOUILLEN - CNRS LABORATOIRE DE BIOGENESE MEMBRANAIRE 0635 : laetitia.fouillen@ubordeaux2.fr
361/381
Mr Thierry FOUQUET - CRP HENRI TUDOR : thierry.fouquet@tudor.lu Miss Sylvie FOURNIER - UMR 5546 CNRS/UPS : fournier@lrsv.ups-tlse.fr Prof Isabelle FOURNIER - UNIVERSIT DE LILLE 1 : isabelle.fournier@univ-lille1.fr Dr Patrick FOURQUET - CNRS : fourquet@ciml.univ-mrs.fr Mr Xavier FRAMBOISIER - LRGP CNRS : xavier.framboisier@ensic.inpl-nancy.fr Dr Julien FRANCK - UNIVERSIT DE LILLE 1 : julien.franck@univ-lille1.fr Dr Yannis FRANCOIS - MATRE DE CONFRENCES UDS : yfrancois@unistra.fr Miss Carine FROMENT - IPBS-CNRS : carine.froment@ipbs.fr
-GMr Guillaume GABANT - INGNIEUR D'TUDES CNRS : guillaume.gabant@cnrs-orleans.fr Miss Fanny GAILLARD - STATION BIOLOGIQUE DE ROSCOFF : gaillard@sb-roscoff.fr Mr Jean-Charles GAILLARD - DSV/IBEB/SBTN : jean-charles.gaillard@cea.fr Dr Patrice GARCIA - L.C.H. : p.garcia@lchfrance.fr Mr Jrme Garin - LABORATOIRE BIOLOGIE GRANDE ECHELLE (BGE) : jerome.garin@cea.fr Dr Jean Luc GATTI - CENTRE DE SOPHIA-ANTIPOLIS : jean-luc.gatti@sophia.inra.fr Mr Mathieu GAUDIN - TECHNOLOGIE SERVIER : mathieu.gaudin@parisdescartes.fr Mr Joseph GAULT - LABORATOIRE DCMR : gault@dcmr.polytechnique.fr Miss Violette GAUTIER - DOCTORANTE : violette.gautier@ipbs.fr Ms Anne-Claude GAVIN - EMBL HEIDELBERG : gavin@embl.de Mr Sylvain GHILINI - GALDERMA R&D : sylvain.ghilini@galderma.com Mrs Franoise GILARD - CNRS IBP : francoise.gilard@u-psud.fr Mr Ludovic GILLET - INSTITUTE OF MOLECULAR SYSTEMS BIOLOGY Mr Rmi GIORDANENGO - RHODIA OPRATION AUBERVILLIERS : remi.giordanengo@eu.rhodia.com Dr Vincent GIRARD - CR1 CNRS : vincent.girard@bayer.com Dr Marion GIROD - UNIVERSIT LYON 1 ET CNRS : mgirod@lasim.univ-lyon1.fr Dr Alexandre GIULIANI - N.A. : alexandre.giuliani@synchrotron-soleil.fr Dr Florence GONNET - Prof : florence.gonnet@univ-evry.fr Dr Anne GONZALEZ DE PEREDO - CNRS : gonzalez@ipbs.fr Mr Yann GOURIOU - CAPM : yann.gouriou@univmed.fr Mr Jrme GOVIN - BIOLOGIE GRANDE ECHELLE U1038 INSERM / CEA / UJF : jerome.govin@cea.fr Mr Samuel GRANJEAUD - INSERM U928 : granjeau@tagc.univ-mrs.fr Mr Seth GRANT - WELLCOME TRUST SANGER INSTITUTE : sg3@sanger.ac.uk Miss Florence GUERARD - CNRS IBP : florence.guerard@u-psud.fr Mr Julien GUERIN - BIOSOLVE CHIMIE : jguerin@biosolve-chemicals.com Mr Philippe GURIN - DSV/IBEB/SBTN : philippe.guerin@cea.fr Mr Vincent GURINEAU - ICSN-CNRS UPR2301 : vincent.guerineau@icsn.cnrs-gif.fr Miss Cline GUIGUE - BERTIN PHARMA : celine.guigue@bertinpharma.com Dr Franois GUILLONNEAU - PLATEFORME PROTOMIQUE UNIVERSIT PARIS DESCARTES francois.guillonneau@parisdescartes.fr Mr Alain GUILLOT - INGENIEUR PROTOMIQUE : alain.guillot@jouy.inra.fr
-H-
Mrs Agns HAGGE - DSV/IBEB/SBTN : agnes.hagege@cea.fr Dr Gregory HAMM - IMABIOTECH : hamm.gregory@imabiotech.com Mr Philippe HAMMANN - PLATEFORME PROTOMIQUE STRASBOURG ESPLANADE : p.hammann@unistra.fr Dr Julie HARDOUIN - UNIVERSIT DE ROUEN : julie.hardouin@univ-rouen.fr Mr Grgoire HARICHAUX - INRA : gregoire.harichaux@tours.inra.fr Mr Fabien HARTEL - STALLERGENES : fhartel@stallergenes.fr Mr Pierre-yves HAUMONT - AB SCIEX : pierre-yves.haumont@absciex.com Dr Michel HBRAUD - DIRECTEUR DE RECHERCHE : michel.hebraud@clermont.inra.fr Miss Sonia HEM - INRA : sonia.hem@supagro.inra.fr Miss Natali HENRIQUES - UNIVERSITE CLAUDE BERNARD LYON 1 : natali.henriques@univ-lyon1.fr Mrs Cline HENRY - MICALIS : celine.henry@jouy.inra.fr Mrs Chirstine HERR - BRUKER DALTONIQUE : christine.herr@bruker.fr Miss Anne-marie HESSE - CEA GRENOBLE : anne-marie.hesse@cea.fr Miss Cline HEU - UNIVERSIT DE FRANCHE COMT - UFR SMP : celine.heu@univ-fcomte.fr
362/381
Miss Mlanie HILLION - LMDF-SME EA 4312 ; UNIVERSIT DE ROUEN : melanie.hillion@hotmail.fr Miss Agns HOVASSE - CNRS IPHC DSA : ahovasse@unistra.fr Mrs Marie HUBERT-ROUX - CNRS UMR 6014, UNIVERSIT DE ROUEN : marie.hubert@univ-rouen.fr Mrs Isabelle HUDE - INSTITUT DE RECHERCHE PIERRE FABRE : isabelle.hude@pierre-fabre.com Dr Hans Caspar HRLIMANN - CNRS IBGC UMR 5095 CNRS *05.56* : hurlimann@ibgc.cnrs.fr Mr Olivier HUTINEL - AB SCIEX : olivier.hutinel@absciex.com
-IMr Gilles IMBERT - CEA/DSV/IBEB/LDCAE : gilles.imbert@cea.fr
-J-
Miss Aurore JAFFUEL - EZUS/ UNIVERSIT CLAUDE BERNARD LYON 1 : aurore_jaffuel@live.fr Mr Emilien JAMIN - INRA AXIOM : jamin@toulouse.inra.fr Miss Lucie JAQUILLARD - DOCTORANTE : lucie.jaquillard@cnrs-orleans.fr Dr Michel JAQUINOD - CHERCHEUR : jaquinod@cea.fr Mr Julien JARDIN - CENTRE DE RENNES : julien.jardin@rennes.inra.fr Miss Nolwenn JARNO - CEA GRENOBLE - BGE -EDYP : nolwenn.jarno@cea.fr Mr Martin JARROLD - CHARGE DETECTION MASS SPECTROMETRY : mfj@indiana.edu Miss Mathilde JOINT - INRA : mathilde.joint@nantes.inra.fr Miss Charlotte JOLY - CENTRE INRA CLERMONT-FD-THEIX : charlotte.joly@clermont.inra.fr Mr Pierre JONLET - UNIVERSIT DE LIGE : pjonlet@ulg.ac.be
-K-
Mr Zied KAABIA - ONIRIS - LABERCA : zied.kaabia@oniris-nantes.fr Dr Said KINANI - EDF-RECHERCHE ET DEVELOPPEMENT : said.kinani@edf.fr Mr Pascal KINTZ - X-PERTISE CONSULTING STRASBOURG : pascal.kintz@wanadoo.fr Miss Agneta KISS - CNRS-DPARTEMENT SERVICE CENTRAL D'ANALYSE : a.kiss@sca.cnrs.fr Dr Thomas KNIGGE - MATRE DE CONFERENCES : thomas.knigge@univ-lehavre.fr Mr Oliver KOHLBACHER - TUEBINGEN UNIVERSITY : oliver.kohlbacher@uni-tuebingen.de Mr Markus KOLBE - DECODON GMBH : kolbe@decodon.com Miss Alexandra KRAUT - CEA GRENOBLE : alexandra.kraut@cea.fr Miss Lauriane KUHN - PLATEFORME PROTOMIQUE STRASBOURG ESPLANADE : ll.kuhn@pansanel.net Mr Akihiko KUSAI - JEOL EUROPE Dr Anthony KWASIBORSKI - PUBLIQUE : akwasiborski@ulg.ac.be
-LMiss Valrie LABAS - INRA : valerie.labas@tours.inra.fr Mr Jrme LACOMBE - CRLC VAL D'AURELLE - IRCM : jerome.lacombe@etu.univ-montp1.fr Miss Anne-sophie LACOSTE - USTL USR 3290 MSAP : anne-sophie.lacoste@univ-lille1.fr Miss Chrystelle LACROIX - IPBS CNRS UMR 5089 UNIVERSIT DE TOULOUSE : chrystelle.lacroix@ipbs.fr Dr Daniel LAFITTE - UNIVERSITE DE LA MEDITERRANEE : daniel.lafitte@univmed.fr Mr Philippe LAGARDE - SANOFI-AVENTIS CHIMIE : philippe.lagarde@sanofi-aventis.com Mr Arnaud LAMARCA - PLATE-FORME BIOPARK D'ARCHAMPS : arnaud.lamarca@agim.eu Miss Marine LAMBERT - INRA : lambert@supagro.inra.fr Mr Pierre-herv LAMBERT - INSTITUT DE RECHERCHES SERVIER : pierre-herve.lambert@fr.netgrs.com Prof Catherine LANGE - INSA DE ROUEN : catherine.lange@univ-rouen.fr Prof Olivier LAPRVOTE - PLATEFORME FINANCIERE PHARMACIE : olivier.laprevote@parisdescartes.fr Dr Colette LARRE - INRA : larre@nantes.inra.fr Mrs Hlne LAVANANT - CNRS UMR 6014 COBRA, UNIVERSIT DE ROUEN : helene.lavanant@univ-rouen.fr Mr Rgis LAVIGNE - SAIC : regis.lavigne@univ-rennes1.fr Miss Adina LAZAR - CRICM UPMC : adina_m31@yahoo.com Prof Bruno LE BIZEC - LABERCA - ONIRIS : bruno.lebizec@oniris-nantes.fr Mr Jean Pierre LE CAER - CNRS ICSN : lecaer@icsn.cnrs-gif.fr Prof Pierre LE MARECHAL - UNIVERSIT PARIS-SUD : pierre.le-marechal@u-psud.fr Miss Diane LEBEAU - CEA-SACLAY : diane.lebeau@cea.fr Miss Melanie LEBRETON - CNRS : lebreton@unistra.fr Dr Rgine LEBRUN - CNRS : rlebrun@ifr88.cnrs-mrs.fr Mr Eric LECLERC - SANOFI-AVENTIS R & D : Eric.Leclerc@sanofi-aventis.com; Marie-Christine.Flipo@sanofiaventis.com
363/381
Mr Alban LECOLLEN - PROTEABIO EUROPE : a.lecollen@eu.proteabio.com Miss Marjorie LEDUC - INSERM U1016 : marjorie.leduc@inserm.fr Ms Jana LEE - PROTEOME SOFTWARE : jana.lee@proteomesoftware.com Mr Bertrand LEGERET - CNRS UMR 6504 : Bertrand.Legeret@univ-bpclermont.fr Mr Thierry LEGOUPIL - THERMO ELECTRON SAS : thierry.legoupil@thermofisher.com Mrs Emmanuelle LEIZE - INSTITUT DE CHIMIE DE STRASBOURG : npotier@chimie.u-strasbg.fr Miss Nadge LEMAHIEU - ANSES DPR UPCMAV : nadege.lemahieu@anses.fr Dr Joel LEMAIRE - LCP : joel.lemaire@u-psud.fr Prof Jrme LEMOINE - EZUS/ UNIVERSIT CLAUDE BERNARD LYON 1 : jerome.lemoine@univ-lyon1.fr Miss Sarah LENNON - CNRS IPHC DSA : Sarah.lennon@etu.unistra.fr Mr Eric LEPOUTRE - GENGAZ EURL : e.lepoutre@gengaz.com Miss Emma-dune LERICHE - CNRS UMR 6014, COBRA, UNIVERSIT DE ROUEN : lericheemmadune@yahoo.fr Mr Eric LEROY - UNIV. PAUL SABATIER : ericlry@free.fr Dr Denis LESAGE - UNIVERSIT PARIS VI : denis.lesage@upmc.fr Mr Mikael LEVI - SHIMADZU : shimadzu@shimadzu.fr Dr Danielle LIBONG - GROUPE DE CHIMIE ANALYTIQUE EA 4041-UPS XI : danielle.libong@u-psud.fr Mrs Sabrina LIGNON - CNRS : lignon@ifr88.cnrs-mrs.fr Dr Martin LINKE - NH DYEAGNOSTICS : m.linke@dyeagnostics.com Dr Laurence LINS - UNIVERSITE DE LIEGE : l.lins@ulg.ac.be Mrs Joanna LIPECKA - INSERM U894 : joanna.lipecka@inserm.fr Dr Damarys LOEW - INSTITUT CURIE : damarys.loew@curie.fr Mr Rmi LONGUESPE - UNIVERSIT DE LILLE 1 : r.longuespee@gmail.com Mr Alain LORPHELIN - DSV/IBEB/SBTN : alain.lorphelin@cea.fr Mrs Corinne LOUTELIER-BOURHIS - CNRS UMR 6014, COBRA, UNIVERSIT DE ROUEN : corinne.loutelier@univrouen.fr Mrs Mathilde LOUWAGIE - CEA : mlouwagie@cea.fr Mr Bernard LYAN - CENTRE INRA CLERMONT-FD-THEIX : bernard.lyan@clermont.inra.fr
-M-
Mr Florian MAIRE - CNRS UMR6014 COBRA, UNIVERSIT DE ROUEN : florian.maire@univ-rouen.fr Mr Yannis MAJOR - UNIVERSITE DE PROVENCE : yannis.major@etu.univ-provence.fr Mr Mohamed MAJOR - UNIVERSITE DE PROVENCE : mohamed.major@etu.univ-provence.fr Mrs Vronique MALARD - DSV/IBEB/SBTN : veronique.malard@cea.fr Mr Christian MALOSSE - CNRS : malosse@dcmr.polytechnique.fr Dr Alain MANG - CRLC VAL D'AURELLE - IRCM : alain.mange@univ-montp1.fr Mr Etienne MAOUT - THERMO ELECTRON SAS : etienne.maout@thermofisher.com Miss Marlne MARCELLIN - INGNIEUR D'TUDES : marlene.marcellin@ipbs.fr Mr Didier MARCELLIN - DSV/IBEB/SBTN : didier.marcellin@cea.fr Dr Christophe MARCHAND - CNRS : christophe.marchand@ibpc.fr Dr Philippe MARIN - IGF : philippe.marin@igf.cnrs.fr Miss Armelle MARTELET - BIOMRIEUX : armelle.martelet@cea.fr Miss Marion MARTIGNAC - UNIV. PAUL SABATIER : marion.martignac@gmail.com Dr Patrice MARTIN - INRA - GNTIQUE ANIMALE ET BIOLOGIE INTGRATIVE : patrice.martin@jouy.inra.fr Mrs Nathalie MARTY-GASSET - INRA / INP : nathalie.martygasset@ensat.fr Dr Laure MARVIN - NESTEC LTD. : laure.marvin-guy@rdls.nestle.com Dr Jean MARY - MATRE DE CONFRENCES : jmary@sb-roscoff.fr Dr Christophe MASSELON - CEA GRENOBLE : christophe.masselon@cea.fr Dr Sabine MATALLANA SURGET - OBSERVATOIRE D'OCANOLOGIE DE BANYULS : sabine.matallana@obsbanyuls.fr Miss Lucrce MATHERON - UPMC - LBM : lucrece.matheron@gmail.com Dr Mariette MATONDO - CONFERENCE : matondo@imsb.biol.ethz.ch Mr Benoit MAUNIT - ENSEIGNANT CHERCHEUR : benoit.maunit@univ-orleans.fr Dr Patrick MAYEUX - INSERM - DRPA05 : patrick.mayeux@inserm.fr Mr Michael MAZARIN - CENTRE DE RECHERCHE DE SOLAIZE - TOTAL : michael.mazarin@total.com Mr Dominique MAZZOCUT - INSERM DRLYS FACTURATION : d.mazzocut@ibcp.fr Dr Thierry MEINNEL - CNRS, ISV-UPR2355 : thierry.meinnel@isv.cnrs-gif.fr Dr Noura MEKLAT - CENTRE INRA CLERMONT-FD-THEIX : Noura.Meklat@clermont.inra.fr
364/381
Mr Sergio MELIS - LECO FRANCE : pascale.rioult@lecofrance.fr Dr Benot MENAND - CNRS : benoit.menand@univmed.fr Mr Franck MERLIER - UTC : merlier@utc.fr Dr Nathalie MESPLET - SANOFI CHIMIE : nathalie.mesplet@sanofi-aventis.com Mr Frdric METRAL - AGILENT TECHNOLOGIES SALES AND SERVICES GMBH AND CO KG : frederic.metral@agilent.com Mrs Emmanuelle MEUDEC - INRA : meudec@supagro.inra.fr Dr Carole MIGN - CENTRE INRA CLERMONT-FD-THEIX : carole.migne@clermont.inra.fr Dr Florence MIGOT-NABIAS - IRD UMR 216 : florence.migot-nabias@ird.fr Mrs Carole MINISINI - ABSCIEX : carole.minisini@absciex.com Dr Guy MIRANDA - INRA-CRJ UNIT GABI_LGS : guy.miranda@jouy.inra.fr Dr Michel- Yves MISTOU - MICALIS : mistou@jouy.inra.fr Dr Catherine MOALI - INSTITUT DE BIOLOGIE ET CHIMIE DES PROTINES : c.moali@ibcp.fr Mrs Laurence MOLINA - SYSDIAG UMR 3145 CNRS BIORAD : laurence.molina@sysdiag.cnrs.fr Mr Jean-marc MONNEUSE - PHYLOGENE : jmmonneuse@phylogene.com Dr Valrie MONNIER - FDRATION DES SCIENCES CHIMIQUES DE MARSEILLE : valerie.monnier@univcezanne.fr Mr Bernard MONSARRAT - CNRS : bernard.monsarrat@ipbs.fr Dr Marie-hlne MONTAN - CNRS : marie-helene.montane@univmed.fr Mr Stphane MOREAU - SHIMADZU : sm@shimadzu.fr Mr Paul MOREL - LABORATOIRE PBS UMR6270 : paul.morel@univ-rouen.fr Miss Claire MOUCHE - INSTITUT DES SCIENCES MOLECULAIRES : c.mouche@ism.u-bordeaux1.fr Mrs Laetitia MOULS - INRA : laetitia.mouls@supagro.inra.fr Mrs Emmanuelle MOUTON BARBOSA - IPBS CNRS : emmanuelle.mouton@ipbs.fr Dr Markus MULLER - SWISS INSTITUTE OF BIOINFORMATICS : markus.mueller@isb-sib.ch Prof Jean-franois MULLER - UNIVERSIT PAUL VERLAINE - METZ : jf.muller66@orange.fr Mr Jean- Pierre MUNIER - JEOL EUROPE SAS : sales@jeol.fr
-N-
Dr Sophie NALLET - CRUCELL SWITZERLAND AG : sophie.nallet@crucell.ch Miss Edlira NANO - INRA : enano@moulon.inra.fr Mr Bertrand NAUDIN - UNIVERSITE DE ROUEN : bertrand.naudin@univ-rouen.fr Mr Alexis NAZABAL - COVALX : alexis.nazabal@covalx.com Mr Luc NEGRONI - CENTRE DE GENOMIQUE FONCTIONNELLE : l.negroni@cbmn.u-bordeaux.fr Mr Claude NETTER - DIONEX : claude.netter@dionex.com Mr Henri NICAR - ABSCIEX : henri-michel.nicar@absciex.com Miss Edith NICOL - CNRS : edith@dcmr.polytechnique.fr
-O-
Mr Antoine OBRY - UMR 6270 : antoine.obry@etu.univ-rouen.fr Miss Alexia ORTIZ - UNIVERSIT DE LILLE 1 - MSAP USR 3290 : alexia.ortiz@ed.univ-lille1.fr Mr Laurent OUERDANE - IPREM UMR 5254 : laurent.ouerdane@univ-pau.fr Mr Sophiane OUGHLIS - UNIVERSIT PARIS 13 CSPBAT LBPS : oughlis.sophiane@yahoo.fr Mr Christopher OVERALL - UNIVERSITY BRISTISH COLUMBIA : chris.overall@ubc.ca
-PMrs Adeline PAGE - IGBMC : adeline.page@igbmc.fr Mrs Fabienne PALGE - F-DBS : fabienne.palge@f-dbs.com Mr Cedric PARIS - ENSAIA : cedric.paris@ensaia.inpl-nancy.fr Dr Xaviera PENNANEC - INRA UMR CSGA : xaviera.pennanec@dijon.inra.fr Dr Marie PROT-TAILLANDIER - IPCM, UNIVERSIT PIERRE & MARIE CURIE PARIS 6 : marie.perot@u-psud.fr Dr Michel PERROT - UNIVERSITE BORDEAUX SEGALEN POLE PROTOMIQUE : michel.perrot@u-bordeaux2.fr Mrs Madeleine PEYTAVIN - THERMO ELECTRON SAS : madeleine.peytavin@thermo.com Dr Delphine PFLIEGER - CHERCHEUSE : delphine.pflieger@univ-evry.fr Mr Olivier PIBLE - DSV/IBEB/SBTN : olivier.pible@cea.fr Miss Carole PICHEREAUX - CNRS : carole.pichereaux@ipbs.fr Ms Paola PICOTTI - ETH ZURICH : paola.picotti@bc.biol.ethz.ch Dr Charles PINEAU - PLATEFORME PROTOMIQUE BIOGENOUEST : charles.pineau@univ-rennes1.fr
365/381
Mr Jrmy PINGUET - CHU GABRIEL MONTPIED : jpinguet@chu-clermontferrand.fr Dr Benot PINSON - CNRS IBGC UMR 5095 CNRS *05.56* : benoit.pinson@ibgc.cnrs.fr Dr Florence POIRIER - UNIVERSIT PARIS 13 : florence.poirier@univ-paris13.fr Mr Pascal PONCET - ESPCI, LSABM : pascal.poncet@espci.fr Dr Frdric PONT - INSERM DRTLS : frederic.pont@inserm.fr Mr Matthieu POPHILLAT - CIML SERVICE DE PROTOMIQUE : pophillat@ciml.univ-mrs.fr Mrs Noelle POTIER - CNRS : npotier@unistra.fr Mr Hugues PREUDHOMME - IPREM UMR 5254 : hugues.preudhomme@univ-pau.fr Dr Michel PRUDENT - SERVICE RGIONAL VAUDOIS DE TRANSFUSION SANGUINE michel.prudent@mavietonsang.ch Mr Cdric PRZYBYLSKI - ETUDIANT : cedric.przybylski@univ-evry.fr Dr Virginie PUECH PAGES - C.N.R.S : puech@lrsv.ups-tlse.fr Dr Estelle PUJOS-GUILLOT - CENTRE INRA CLERMONT-FD-THEIX : estelle.pujos@clermont.inra.fr
-Q-
Miss Lei QI - DSV/IBEB/SBTN : ley.qi@cea.fr Mr Jusal QUANICO - MALDI IMAGING TEAM (FABMS) : jusal.quanico@etudiant.univ-lille1.fr Mrs Florence QULIN - UFR DES SCIENCES DE LA SANT PIFO . UPRES EA 2493 : florence.quelin@uvsq.fr
-RMiss Amandine RACAUD - CENTRE DE RECHERCHE DE SOLAIZE - TOTAL : amandine.racaud@total.com Mr Nicolas RAYNAL - CEM WAVES : nicolas.raynal@cem.com Dr Virginie REDEKER - LEBS, CNRS : redeker@lebs.cnrs-gif.fr Miss Sandra RENIER - DOCTORAT : sandra.renier@clermont.inra.fr Dr Marie-france ROBBE - CEA : marie-france.robbe@cea.fr Miss Ophlie ROBINE - CNRS : ophelie@dcmr.polytechnique.fr Mr Gislain ROCHEBLOINE - SANOFI : gislain.rochebloine@sanofi.com Dr Beatrice ROCHER - LEMA EA32 22, UFR ST : beatrice.rocher@univ-lehavre.fr Mr David RODARIE - TECAN FRANCE SAS : david.rodarie@tecan.com Mrs Valrie ROFIDAL - INRA : rofidal@supagro.inra.fr Dr Hlne ROGNIAUX - INRA PLATE-FORME BIBS : helene.rogniaux@nantes.inra.fr Mr Christian ROLANDO - UNIVERSITE DE LILLE : christian.rolando@univ-lille1.fr Mr David ROPARTZ - INRA : david.ropartz@nantes.inra.fr Mrs Florence ROUX-DALVAI - IPBS-CNRS : florence.dalvai@ipbs.fr Miss Carolina RUBIANO - PONTIFICIA UNIVERSIDAD JAVERIANA : carolinarubiano1983@gmail.com Mr Frank RUFFENACH - IGBMC : rf4@igbmc.fr
-SDr Emmanuelle SACHON - UPMC - LBM : emmanuelle.sachon@upmc.fr Dr Sandrine SAGAN - UPMC - LBM : sandrine.sagan@upmc.fr Mrs Nicole SAGE - DSV/IBEB/SBTN : nicole.sage@cea.fr Miss Laila SAGO - INSERM - DRPA05 : laila.sago@inserm.fr Miss Virginie SALNOT - INSERM DRPA05 : virginie.salnot@parisdescartes.fr Mr Thierry SALOMON - CELL BIOSCIENCES : tsalomon@cellbiosciences.com Dr Jean- Yves SALPIN - UNIVERSIT D'EVRY VAL D'ESSONNE : jean-yves.salpin@univ-evry.fr Prof Arnaud SALVADOR - EZUS/ UNIVERSIT CLAUDE BERNARD LYON 1 : arnaud.salvador@univ-lyon1.fr Dr Nicolas SALVETAT - SYSDIAG UMR3145 CNRS/BIO-RAD : nicolas.salvetat@sysdiag.cnrs.fr Prof Michel SALZET - UNIVERSIT DE LILLE 1 : michel.salzet@univ-lille1.fr Mr Xavier SAUNIER - TECAN FRANCE SAS : xavier.saunier@tecan.com Mrs Marcelle SAUZON - BIOMRIEUX : marcelle.sauzon@biomerieux.com Mr Thierry SAYD - INGENIEUR : thierry.sayd@clermont.inra.fr Mrs Christine SCHAEFFER-REISS - CNRS IPHC DSA : christine.schaeffer@unistra.fr Mr Jean- Louis SCHAFFAR - EURISO-TOP : jlschaffar@eurisotop.com Mrs Therese SCHEMBRI - UNIVERSITE DE LA MEDITERRANEE : therese.schembri@univmed.fr Dr Odile SCHILTZ - IPBS, CNRS : odile.schiltz@ipbs.fr Prof Jean- Marie SCHMITTER - UNIVERSIT DE BORDEAUX : jm.schmitter@cbmn.u-bordeaux.fr Mr Sbastien SCHRAMM - LSMCL - UNIVERSIT PAUL VERLAINE : schramm@univ-metz.fr Miss Aurelie SEASSAU - CENTRE DE SOPHIA ANTIPOLIS INRA : aurelie.seassau@sophia.inra.fr
366/381
Mr Gatan SEEBURN - EURISO-TOP : gseeburn@eurisotop.com Miss Lyna SELLAMI - PIT2/CRO2 : lina.sellami@hotmail.com Mr Etienne SMON - INRA UMR CSGA : Etienne.Semon@dijon.inra.fr Mrs Hlne SENECHAL - ESPCI, LSABM : helene.senechal@espci.fr Prof Michel SEVE - PLATEFORME PROTOMIQUE PROMTHE : Michel.Seve@ujf-grenoble.fr Mr Martial SEVENO - IR CNRS PLATE-FORME DE PROTOMIQUE FONCTIONNELLE : martial.seveno@igf.cnrs.fr Dr Pascal SEYER - CNRS : pascal.seyer@igf.cnrs.fr Mr Alexandre SEYER - CEA SACLAY : alexandre.seyer@cea.fr Mr Youcef SHAHALI - ESPCI, LSABM : youcef.shahali@espci.fr Miss Estelle SIBILLE - LSMCL - UNIVERSIT PAUL VERLAINE : sibille@univ-metz.fr Dr Luca SIGNOR - INSTITUT DE BIOLOGIE STRUCTURALE : luca.signor@ibs.fr Mr Romain SIMON - EZUS/ UNIVERSIT CLAUDE BERNARD LYON 1 : rom1.simon@orange.fr Mr Gilbert SKORSKI - PHYLOGENE : g.skorski@phylogene.com Dr Audrey SOLGADI - SAMM - FACULT DE PHARMACIE - UPS 11 : audrey.solgadi@u-psud.fr Mr Jean Marc SOUQUET - INRA : souquet@supagro.inra.fr Dr Jonathan STAUBER - IMABIOTECH : stauber.jonathan@imabiotech.com Mr Alexandre STELLA - CNRS : alexandre.stella@ipbs.fr
-T-
Prof Jean- Claude TABET - IPMC-UMR 7201-UFR726-LCSOB-CASE 45 : jean-claude.tabet@upmc.fr Mr Mhdi TAIYA - UBO-UNIVERSIT DE BRETAGNE OCCIDENTALE : mehdi.taiya@univ-brest.fr Dr Marianne TARDIF - CEA : marianne.tardif@cea.fr Mrs Ana Paula TEIXEIRA-GOMES - INRA : teixeira@tours.inra.fr Dr Nathan TENE - CENTRE UNIVERSITAIRE DE FORMATION ET DE RECHERCHE : nathan.tene@univ-jfc.fr Miss Laetitia THERON - INRA - ENSAT : laetitia.theron@ensat.fr Mrs Danile THIERSE - CNRS IPHC DSA : dthierse@unistra.fr Dr Robert Alain TOILLON - SERVICE FACTURIER : robert.toillon@univ-lille1.fr Dr David TOUBOUL - ICSN CNRS UPR2301 : touboul@icsn.cnrs-gif.fr Mr Mathieu TRAUCHESSEC - CEA / METABOLIC EXPLORER : mathieu.trauchessec@cea.fr Miss Sarah TRIBOULET - ETUDIANTE EN THSE : sarah.triboulet@gmail.com Mr Yury TSYBIN - EPFL LAUSANNE : yury.tsybin@epfl.ch
-U-V-
Dr Svetlana UZBEKOVA - INRA : svetlana.uzbekova@tours.inra.fr Dr Gilles VALETTE - UNIVERSIT DE MONTPELLIER 2 : gilles.valette@univ-montp2.fr Mr Cyrille VALLE - ONIRIS - LABERCA : cyrille.vallee@oniris-nantes.fr Dr Benoit VALOT - PAPPSO, UMR DE GNTIQUE VGTALE : valot@moulon.inra.fr Mr Gerard VAN DER LAAN - MS VISION BV : gl@msvision.eu Mr Guillaume VAN DER REST - CNRS : gvdr@dcmr.polytechnique.fr Prof Alain VAN DORSSELAER - CNRS IPHC DSA : vandors@unistra.fr Dr Franck VANDERMOERE - INSTITUT DE GNOMIQUE FONCTIONNELLE : franck.vandermoere@igf.cnrs.fr Dr Anne-sophie VERCOUTTER-EDOUART - UNIT GLYCOBIOLOGIE STRUCTURALE ET FONCTIONNELLE : annesophie.vercoutter@univ-lille1.fr Dr Yann VERDIER - ESPCI PARISTECH : yann.verdier@espci.fr Mr Patrick VESCOVI - LGS LAB GAZ SYSTEMS : patrick.vescovi@labgaz.fr Mr Didier VIALA - ASSISTANT INGNIEUR : didier.viala@clermont.inra.fr Mr Frdric VIART - ASA - ADVANCED SOLUTIONS ACCELERATOR : fviart@asa-sas.com Mr Claude VIDAUD - DSV/IBEB/SBTN : claude.vidaud@cea.fr Dr Sbastien VILAIN - UNIVERSITE BORDEAUX SEGALEN POLE PROTOMIQUE : sebastien.vilain@ubordeaux2.fr Mr Claude VILLARD - UNIVERSITE DE LA MEDITERRANEE : claude.villard@univmed.fr Mr Jean- Baptiste VINCENDET - AB SCIEX : jean-baptiste.vincendet@absciex.com Mrs Jolle VINH - ESPCI PARISTECH : joelle.vinh@espci.fr Ms Olga VITEK - PURDUE UNIVERSITY : ovitek@purdue.edu Mrs Christiane VITRY - INSTITUT DES SCIENCES MOLECULAIRES : c.vitry@ism.u-bordeaux1.fr Mr Kees VLAK - HARLOW ENTERPRISE HUB : kvlak@advion.com
367/381
-WDr Reiner WESTERMEIER - SERVA ELECTROPHORESIS GMBH : reiner.westermeier@serva.de Mr Philippe WOLFF - PLATEFORME PROTOMIQUE STRASBOURG ESPLANADE : p.wolff@ibmc-cnrs.unistra.fr Mr Nico WORTEL - MS VISION BV : gl@msvision.eu Miss Jianqing WU - CNRS : wu@dcmr.polytechnique.fr
-ZMr Affif ZACCARIA - INSERM U836-EQ7 : zaccaria.affif@gmail.com Mrs Isabelle ZANELLA-CLEON - INSERM DRLYS FACTURATION : i.zanella-cleon@ibcp.fr Ms Zita ZENDONG - ONIRIS - LABERCA : zita.zendong@oniris-nantes.fr Miss Anna ZIEMIANIN - CNRS UMR 6014, COBRA, UNIVERSIT DE ROUEN : annaziemianin@gmail.com
368/381
Author Index
369/381
-AABDEL-WAHAB, Yasser H.A....................................222 ABEILHOU, Pierre...................................................169 ABID, Salwa.............................................................93 ABSALON, Christelle...............................268, 269, 358 ADAM, Claire.................................................305, 358 ADRIANO, Jean-Marc.............................................101 AEBERSOLD, Ruedi.................................................277 AFONSO, Carlos...24, 80, 190, 225, 280, 303, 320, 358 AHRNE, Erik.............................................................71 AK, Francine.........................................................129 AKLOUL, Rym.........................................................219 AL ALI, Ahmad................................................131, 358 ALAYI, Tchilabalo...................................................248 ALBERTIN, Warren.................................................261 ALBIGOT, Renaud...........................................252, 358 ALBRIEUX, Florian..................................176, 192, 358 ALEXANDER CHRISTIE-OLEZA, Joseph....................306 ALEXANDROV, Theodore.......................................232 ALKHALFIOUI, Fatima...............................................91 ALLAIN, Franois............................................315, 358 ALLEGRAND, Julie....................................................53 ALLORY, Yves...........................................................85 ALMEIDA, Reinaldo.........................281, 299, 318, 322 ALMERAS, Lionel............................................227, 358 ALVES, Sandra................................144, 205, 225, 320 AMOUYEL, Philippe................................................138 ANDR, Patrice......................................................142 ANNE CREMER , Galle..........................................153 ANNE LORIOT, Marie.............................................245 ANSELME, Michel...........................................345, 346 ANTIGNAC, Jean-Philippe.................57, 137, 221, 247 ANTOINE, Rodolphe....54, 61, 133, 154, 184, 210, 280 ARAFAH, K.............................................................302 ARC, Erwann..........................................................278 ARGENTINI, Manuela.............................................158 ARMENGAUD, Jean 8, 14, 79, 283, 291, 306, 314, 315, 316, 358 ARMENTROUT, Peter B..........................................303 ARNAUDGUILHEM, Carine.............................194, 358 ARVERS, P..............................................................302 ASTRUC, Thierry.....................................................170 ATMANENE, Cedric................................................292 ATTEIA, Ariane.......................................................224 AUBERT, Lo..................................................150, 358 AUBOURG, Patrick.................................................193 AUBRIET, Frdric............................................62, 358 AUBRY, Laurence...................................................304 AUDE-GARCIA, Catherine.........................................88 AUDEBERT, Stphane.............................227, 229, 266 AUDOUARD-COMBE, Fabien..................................214 AUDRAN, Emilie.....................................................312 AURAND, Rmy..............................................278, 358 AUVYNET, Constance.......................................68, 187 AUZEIL, Nicolas..............................................185, 193 AVERSENG, Olivier.................................................211
AYCIRIEX, Sophie............................185, 193, 245, 358 AYMONIER, Cyril....................................................269 AZANDO, E. V. B.....................................................235 AZOULAY, Michel...................................................182 AZRIA, David............................................................96
-B-
BACHA, Hassen........................................................93 BAENA, Sandra......................................................291 BAGREL, Denyse....................................................180 BAILLET, Arlette.....................................................320 BAILLY-CHOURIBERRY, Ludovic................................81 BAKKALI, Nawal.....................................................227 BALAKRISHNAN, Indran.........................................126 BALLIAS, Patricia....................................................253 BALLOT, Eric..........................................................284 BANA, Emilie..........................................................180 BAPTISTE FOURNIER, Jean.....................................124 BARBIER, Pascale...................................................309 BARNES, Alan.................................................196, 197 BARRRE, Caroline.................................141, 200, 358 BARSAN, Cristina...................................................336 BARTHE, Christophe..............................................127 BARTHE, Damien................................8, 101, 186, 358 BARYLYUK, Konstantin.............................................50 BASCANDS, Jean-Loup...........................................260 BAUDELET, Emilie...................................227, 266, 358 BAUDET, Mathieu...............................8, 101, 267, 358 BAUDIN, Bruno..............................................117, 358 BAUDIN, Karima.......................................19, 275, 358 BAUDOT, Robert............................................326, 333 BAUDOUIN, Christophe..........................140, 159, 340 BAUMGART, Jan....................................................281 BAUTISTA-ORTN, Ana-Beln.................................112 BAUVAIS, Cla........................................................339 BEAU, Mathilde................................65, 172, 182, 358 BEAUFOUR, Martine......................................335, 358 BEAUNE, Philippe...........................................131, 245 BECAMEL, Carine.....................................................48 BCARD, Guillaume.................................................90 BECCHI, Michel........................................................69 BECHARA, Chrine.........................................206, 358 BECHER, Franois......75, 118, 144, 164, 181, 301, 358 BECKER , Emmanuelle......................................72, 230 BECUE, Thierry.........................................................67 BEISSON, Fred........................................................101 BELEOKEN, Elvire...................................................284 BELGACEM, Omar...........................126, 128, 264, 309 BELGHAZI, Maya............................................204, 358 BELGHIT, Hanane.....................................................80 BELLE, Valrie........................................................350 BELLINA, Bruno......................................................184 BELOTTI, Edwige....................................................266 BEN AMEUR, Randa...............................................311 BNARD, Camille...................................................253 BNO, Nolle.........................................................175 BENOIT-MARQUI, Florence..................................147
370/381
BENSAID, Fethi......................................................265 BENSLIMANE-AHMIM, Zahia..................................228 BENZ, Nathalie.......................................................338 BERGER, Franois...........................................302, 313 BERNARD-SAVARY, Pierre..............................354, 355 BERNILLON, Stphane....................................253, 359 BERQUAND, Alexandre..........................................325 BERTHON, Laurence.......................................349, 359 BERTILE, Fabrice.......................................77, 294, 359 BERTIN, Denis................................................200, 201 BERTIN, Gwladys............................................160, 359 BETTAIEB, Ali.........................................................254 BIACCHI, Michael...................................273, 347, 359 BIAIS, Benoit..........................................................253 BIAN, Wanping......................................................336 BIANCHI, Leonardo................................................352 BIARC, Jordane...........................................25, 49, 359 BICH, Claudia........................22, 52, 94, 240, 324, 359 BICHON, Emmanuelle............................................247 BIENVENUT, Willy V....................24, 63, 162, 163, 359 BIERLA, Katarzyna..................................................194 BIGEARD, Jean.......................................................157 BILLON, Emmanuelle.............................................101 BLANCHET, Sandra.................................................158 BLAND, Cline............................................8, 316, 359 BLEIN-NICOLAS, Mlisande....................261, 263, 359 BLESBOIS, Elisabeth...............................................114 BLINET, Sylvie........................................................323 BOCKAERT, Jol....................................48, 76, 97, 359 BODGE, Annegret..................................................337 BODIN, Audrey...............................................169, 359 BOERI ERBA, Elisabetta..............................24, 50, 359 BOIREAU, Wilfrid....................................255, 307, 359 BOISSEL, Pierre......................................................330 BOISSON-VIDAL, Catherine....................................228 BOLBACH, Grard.....................68, 187, 217, 231, 359 BOLLOTTE, F..........................................................134 BOLOT, Stphanie..................................................109 BOLVIN, Capucine..................................................311 BONHOMME, Ludovic......................................83, 263 BONNAFF, David..................................................105 BONNAIRE, Yves.......................................81, 122, 328 BONNEL, David........................................................73 BONNEU, Marc.............9, 74, 127, 188, 226, 243, 359 BORG, Jean-Paul....................................................266 BORNARD, Isabelle................................................207 BORNERT, Olivier.....................................................91 BOSSELUT, Nelly....................................................117 BOTH, Jean-Pierre..................................................130 BOUAMRANI, Ali............................................313, 359 BOUCHONNET, Stphane...........................25, 59, 359 BOUKENOUCHE, Asma...........................................219 BOUKHERROUB, Rabah............................................64 BOULAY, Mylne....................................................151 BOUQUET, Valentin..................................19, 209, 359 BOUR, Jrme.........................................................70
BOURCIER, Sophie...................................................59 BOURDERIOUX, Matthieu......................................342 BOURDON, Valrie.........................................265, 359 BOURGOGNE, Emmanuel...............................108, 359 BOURGOIN-VOILLARD, Sandrine............................303 BOURGUIGNON, Jacques.......................................331 BOURHIS, Jean-Henry............................................284 BOURMAUD, Adle................................................212 BOURSIER, Cline...................................174, 278, 359 BOUSQUET-DUBOUCH, Marie-Pierre.......60, 277, 359 BOUTET, Isabelle...................................................339 BOUYSSI, David...............65, 109, 260, 289, 336, 359 BOUZAIDI CHEIKHI, Imad.........................................74 BOUZOM, Franois..................................................73 BOYON, Charlotte..................................................276 BRADSHAW, Ralph...................................................49 BRAGUER , Diane...................................................282 BRAHIM, Bessem....................................205, 225, 359 BRASME, Bernard..................................................107 BRAY, Fabrice........................................................233 BRETONNIERE, Yann................................................54 BREUIL, Benjamin..................................................260 BRIGNOLE-BAUDOUIN, Franoise..........140, 159, 340 BRIZARD, Jean-Paul................................................195 BROUSSARD, Cdric.................................95, 146, 244 BROUSSOLLE, Vronique.......................................207 BROYER, Michel.....................................................184 BRUGIRE, Sabine.......................8, 224, 236, 304, 359 BRULEY, Christophe. 8, 85, 98, 99, 102, 186, 224, 236, 242, 267, 304, 305, 331, 332, 359 BRUN, Virginie........................26, 32, 98, 99, 102, 359 BRUNEAUX, Matthieux..........................................198 BRUNEEL, Arnaud..................................................117 BRUNEL, Luc............................................................55 BRUNELLE, Alain...52, 53, 94, 131, 139, 140, 159, 170, 185, 240, 340 BRUNET, Claire.......................................133, 210, 359 BUCHMANN, William.............................115, 300, 359 BUCHRIESER, Carmen............................................145 BUE, Luc...............................................................327 BUISSON, Corine......................................................82 BULET, Philippe..............................................295, 359 BULET , Audrey....................................................326 BULINGAME, Al........................................................49 BULLET, P...............................................................302 BULTELLE, Florence................................................274 BUNEL, C................................................................353 BUR, Corinne............................................25, 51, 359 BUREL, Alexandre............................................77, 359 BURLET-SCHILTZ, Odile......60, 65, 109, 143, 172, 182, 183, 246, 250, 259, 260, 277, 289, 319 BURNOUF, Dominique Y........................................171 BUSNEL, Jean-Marc...............................................273
-C-
CABASSON, Ccile..................................................253 CABATON, Nicolas...................................................56
371/381
CACAS, Jean-Luc......................................................51 CADENE, Martine...................................271, 335, 359 CALLIGARIS, David....................................78, 282, 359 CALVO, Florent......................................................192 CAMADRO, Jean-Michel..........................................84 CAMOIN, Luc............................93, 227, 229, 266, 359 CANLET, Ccile.........................................................56 CANON, Francis..............................133, 293, 317, 359 CANTEL, Sonia.......................25, 55, 64, 136, 173, 359 CAPOMACCIO, Robin...............................................69 CARAPITO, Christine...........................26, 77, 312, 359 CARR, Vincent................................................62, 359 CARRIER, Marion...................................................269 CARRILLO , Stphanie............................................112 CARTIER, Nathalie..................................................193 CASSIER-CHAUVAT, Corinne..................................166 CAST, Delphine......................................................104 CASTAING , Bertrand.............................................271 CASTEL, Patricia.............................................268, 359 CATHERINE FONBONNE, ...............................138, 361 CAUBET, Ccile......................................................260 CAZALS, Guillaume.........................................241, 359 CECCHELLI, Romo................................................203 CENTENO, Delphine.......................................324, 360 CERRUTI, Christopher....................................170, 360 CHAFEY, Philippe........................89, 95, 146, 161, 360 CHAFSEY, Ingrid.....................................................178 CHAIMBAULT, Patrick.............................142, 180, 360 CHALKLEY, Robert....................................................49 CHALMEL, Frdric................................................239 CHAMBERS, Matthew............................................289 CHAMBERT, Stephane.............................................54 CHAMBON, Christophe...178, 218, 262, 323, 324, 360 CHAMINADE, Pierre...............................................320 CHAMOT-ROOKE, Julia............9, 58, 84, 120, 148, 360 CHAN TCHI SONG , Philippe...................................177 CHAN, Philippe...............................................177, 212 CHANGOTADE, S....................................................134 CHAOUI, Karima.....................................250, 277, 360 CHAPEL, Agns.......................................................304 CHARDAN, Laetitia.................................................283 CHARDONNET, Solenne.................................166, 360 CHARLES, Laurence...70, 141, 200, 201, 202, 210, 360 CHARLEUX, Bernadette..........................................154 CHARPIN, Denis.....................................................161 CHARRETIER, Yannick.................................24, 66, 360 CHARRIER, Jean-Philippe..................................66, 138 CHASTANT-MAILLARD, Sylvie.................................156 CHATELLIER, Sonia...................................................66 CHATTI GAZZAH, Amel.............................................93 CHAUMONT-DUBEL, Sverine.................................97 CHAUMOT, Arnaud................................................214 CHAUSSINAND, Guylaine...............................306, 360 CHENAU, Jrme........................................24, 75, 360 CHENDO, Christophe..............................141, 200, 360 CHREAU, Sylvain.......................................26, 57, 360
CHERRAD, Semcheddine........................................251 CHEVET, Eric..........................................................243 CHEVOLLEAU, Sylvie......................................137, 360 CHEYNIER, Vronique....................................119, 317 CHHUON, Cerina............................................342, 360 CHIAPPETTA, Giovanni...........................296, 310, 360 CHIARA GUERRERA, Ida.........................................342 CHIROT, Fabien..............................190, 191, 192, 360 CHNEIWEISS, Herv...............................................312 CHOLLET-MARTIN, Sylvie.......................................161 CHORT, Alice..........................................................245 CHRISTIE-OLEZA, Joseph A.....................................316 CLAIR, Grmy...........................................22, 79, 360 CLAPAROLS, Catherine...........................147, 265, 360 CLARY, Guilhem...............................................95, 146 CLAUDE TABET, Jean..............................................320 CLAUDE, Emmanuelle............................................348 CLAVEROL, Stphane........74, 127, 188, 226, 243, 360 CLAVERYS, Jean-Pierre...........................................259 CLAVREUL, Anne....................................................238 CLMENON, Benjamin.........................................135 CLOTTES, Eric.........................................................319 COADOU, Gael.......................................................152 COCQUEREZ, Thophile.........................................155 COFFINIER, Yannick..................................................64 COLIN, Florent.........................................................92 COLINET, Dominique.............................................204 COLLARD, Alfred....................................................216 COLLIGNON, Anne.................................................174 COLLIN-FAURE, Vronique.......................................88 COM, Emmanuelle.................................238, 239, 360 COMPAGNON, Isabelle..........................................184 CONREUX, Alexandra.............................................248 CONTREPOIS, Kvin...............................................167 COQUET, Laurent...................................212, 222, 360 CORBET, Cyril.................................................150, 360 CORMANT, Florence................................................81 CORNU, David................................................158, 360 COSETTE, Pascal.....................................177, 212, 360 COSTAGLIOLI , Patricia...........................................127 COTTON, Anthony.................................................226 COUDERC, Rachel..................................................295 COUILLAULT, Carole...............................................100 COULON, Maxime..................................................110 COURANT, Frdrique.............................................57 COURCOUL, Mlaine......................185, 193, 245, 360 COUREUX, Pierre-Damien......................................297 COURNAC, Laurent................................................224 COURT, Magali.................................................85, 305 COUT, Yohann......................102, 186, 267, 332, 360 COUTOULY, Marie-Aude........................................215 COUVET, Morgane.................................220, 278, 360 CRAVELLO, Laetitia................................................118 CREN-OLIV, Ccile...........................82, 326, 333, 334 CRONE, Catharina..................................................116 CROUZET, Marc.....................................................127
372/381
CUIN, Stphan.....................................................101 CULARD, Franoise................................................271 CUNY, Celine............................................................91 CUNY, Grard........................................................195
-DDAGOSTO, Franck.................................................154 DACHEUX , Jean-Louis............................................239 DAGANY, Xavier.......................................................61 DAGHER, Zeina......................................................199 DAHMANI, Abdelkader..........................................241 DAIGNAN-FORNIER, Bertrand..................................74 DALLONGEVILLE, Sophie........................................258 DAMOC, Eugen......................................................116 DANIEL, Rgis.................................................105, 115 DARBOUR, Florence.................................................54 DAROLLES, Carine..........................................283, 360 DARROUZET, Elisabeth..........................................314 DAUGHERTY , Sean................................................275 DAULY, Claire.................................................116, 360 DAUTREY, Sbastien..............................................120 DAY, Robert...........................................................276 DE HAAN, Bjorn.....................................................286 DE LA PORTE, Sabine..........................................52, 94 DE PAU, Edwin...............................................216, 219 DE PAUW, Edwin...................................................216 DE VIENNE, Dominique..........................................261 DE WAZIERS, Isabelle.............................................245 DEBORDE, Catherine.............................................253 DEBRAUWER, Laurent......................56, 123, 137, 361 DEBUIRE, Jacques..................................................147 DECEUNINCK, Yoann......................................247, 361 DECHAVANNE, Clia.................................89, 321, 361 DECOTTIGNIES, Paulette................................166, 361 DECOURCELLE, Mathilde................................308, 361 DECROP, Wim........................................................285 DEFER, Christine............................................233, 234 DEFFIEUX, Alain.....................................................268 DEKKER, Karsten....................................................286 DELANGHE, Bernard..............................................116 DELAUNOIS, Bertrand............................................278 DELBOS, Jean-Marie................................................73 DELCOURT, Nicolas..................................65, 172, 182 DELMAS, Agns......................................................153 DELOLME, Frdric..........................................69, 361 DELORON, Philippe................................................160 DELSUC, Marc-Andr.......................................44, 215 DELVOLV, Alice....................................................225 DEMETTRE, Edith...........................................195, 361 DEMEY-THOMAS, Emmanuelle..............310, 327, 339 DENARI, Jean.........................................................90 DENERY-PAPINI, Sandra.........................................103 DENIER, Xavier.......................................................274 DENISOV, Eduard...................................................116 DPREZ, Benoit........................................................73 DERACHE, Chrystelle..............................................110 DERACINOIS, Barbara.....................................203, 278
DERNIS, Dominique........................................233, 234 DERVILLY-PINEL, Gaud.....................................57, 122 DESBENOIT, Nicolas................140, 159, 240, 340, 361 DESMAZIRES, Bernard..........................................300 DESMONS, Annie...................................................276 DESTANDAU, Emilie...............................................125 DESVAUX, Mickal.........................................178, 262 DEUTSCHER, Josef.................................................129 DEWAELE, Laure....................................................189 DIALLO, Dvy...........................................................88 DIAS ABELAIRAS, Helia...........................................256 DIEBATE, Oumar....................................................217 DIEMER, Hlne.......................................................88 DIERYCKX, Cindy............................................251, 361 DIEUSET, Gabriel....................................................232 DIOLOGENT, Laurent......................................231, 361 DIOUF, Ibrahima....................................................227 DIRAT, Batrice......................................................250 DITTRICH-DOMERGUE, Franziska...........................106 DJELTI, Fathia.........................................................193 DJIDJA, Marie-Claude............................................348 DOIZI, Denis...........................................................124 DOMERGUE, Frdric......................................51, 106 DOMINICI, Christian...............................................202 DOMON, Bruno............................24, 36, 85, 288, 361 DONFACK, Bryl.......................................................94 DOUSSINEAU, Tristan............................................154 DRIDI, Imene...........................................................63 DRUART, Xavier..............................................114, 189 DUBAN-DEWEER, Sophie.......................................203 DUBOIS, Marc..........................................................56 DUBRULLE, Marie-Pierre........................................252 DUCHAMP, Christian......................................176, 361 DUCHATEAU, Magalie....................................145, 361 DUCLOS-VALLE, Jean-Charles...............................284 DUCOROY, Patrick..................254, 255, 307, 325, 361 DUCOUX-PETIT, Manuelle..............................319, 361 DUGOUJON, Jean-Michel.........................................89 DUGOURD, Philippe.......9, 54, 61, 133, 154, 184, 190, 191, 192, 210, 213, 280, 361 DUMAS, Philippe...................................................171 DUMNIL, Guillaume...............................................84 DUPIERRIS, Vronique................................8, 305, 361 DUPORT, Catherine.................................................79 DUPR, Mathieu.........................23, 64, 136, 173, 361 DUPUIS, Alain........................................................102 DURAND, Fabrice...................................................274 DURAND, Sophie...................................................221 DURAND, Sylvere...................................................118 DURIEZ, Elodie.........................................85, 288, 361 DURIGHELLO, Emie........................................316, 361 DUYCKAERTS, Charles....................................185, 240
-EEDDARKAOUI, Sabiha.............................................327 EDELMAN, Aleksander...................................342, 361 EDWARDS-JONES, Valery.......................................126
373/381
EGEA, Isabel...........................................................336 EIKEL, Daniel..................................................318, 322 EJSING, Christer......................................281, 318, 322 EL OSTA, Marven...........................................307, 361 ELFAKIR, Claire.......................................................125 ELIE-CAILLE, Cline.................................................325 EMADALI, Anouk...................................................305 ENJALBAL, Christine............55, 64, 136, 173, 241, 361 ENJALBERT, Quentin...................23, 54, 213, 280, 361 ERARD, Monique...................................................319 ESCUDIER, Bernard................................................342 ESPAGNE, Christelle.........................................63, 163 ESPAGNON, Isabelle..............................................130 ESTOUR, Franois..................................................257 EVRARD , Bertrand...........................................72, 230 EWBANK, Jonathan................................................100 EZAN, Eric.................................75, 164, 167, 301, 361
-F-
FABRE, Bertrand.........................................22, 60, 361 FABRE, N................................................................235 FANTON, Laure......................................................142 FEDERICI, Christian........................................146, 244 FENAILLE, Franois....................75, 144, 167, 301, 361 FEREC, Claude........................................................338 FERNANDEZ, Bernard.....................................316, 361 FERNANDEZ, Xavier...............................................218 FERNIE Alisdair......................................................253 FERRO, Myriam...8, 101, 224, 236, 242, 305, 332, 361 FERRU, Geoffroy....................................................349 FIEVET, Nadine......................................................160 FILDIER, Aurlie.......................................................82 FLAHAUT, Christophe.....................................203, 361 FLAMENT-WATON, Marie-Magdeleine....................82 FLATT, Peter R.......................................................222 FLEURY, Cline...........................................8, 332, 361 FLIS, Paulina...........................................................329 FLORENT, Jean-Claude...........................................182 FOCSA, Cristian......................................................231 FONTAINE, Albin....................................................227 FOREST, Eric...................................................135, 361 FOUILLEN, Laetitia.........................................106, 361 FOUQUET, Thierry......................................25, 70, 362 FOURNIER, Franoise.............................................303 FOURNIER, Isabelle.........140, 231, 276, 340, 351, 362 FOURQUET, Patrick........................................100, 362 FRAMBOISIER, Xavier.....................................343, 362 FRANCESCHI, Christine............................................66 FRANCK, Julien...............................................351, 362 FRANOIS, Yannis...........................272, 273, 347, 362 FRDRIC CARLIN, .................................................207 FROMENT, Carine..........................................246, 362 FROMENTY, Galle................................................173 FROTTIN, Frdric....................................................63 FULCRAND, Hlne................................................112
-G-
GABANT, Guillaume.......................................271, 362 GABELLE, Audrey...................................................181 GAIGEOT, Marie-Pierre..........................................208 GAILLARD, Jean Charles.........................................283 GAILLARD, Jean-Charles.....................8, 314, 316, 362 GAL, Olivier............................................................223 GALLAGHER-GAMBARELLI, Maighread..................267 GALLIEN, Sbastien................................................288 GARBAY, Bertrand..........................................127, 226 GARCIA, Patrice..................................22, 81, 328, 362 GARIN, Jrme 8, 9, 14, 85, 98, 99, 102, 242, 331, 362 GARNIER, Nicolas...................................................258 GARRIC, Jeanne..............................................214, 326 GATELLIER, Philippe...............................................323 GATTI, Jean-Luc.....................................................204 GAUDIN, Karen........................................................51 GAUDIN, Mathieu..................................185, 193, 362 GAULT, Joseph...........................................23, 84, 362 GAUTHIER, Sbastien.............................................169 GAUTIER, Hlne...................................................253 GAUTIER, Violette.............65, 143, 172, 182, 183, 362 GAVALDA, Sabine..................................................344 GAVOILLE, Joseph..................................................255 GEAIRON, Audrey..................................................103 GEFFARD, Olivier...................................................214 GEIGER, Sophie......................................................243 GNARD, Michel....................................................253 GENIN, Eric............................................................344 GENTY, Christophe..................................................59 GRARD, Nadine............................................114, 189 GERVASI, Gaspard............................................66, 164 GIBON, Yves...........................................................253 GIGLIONE, Carmela..................................63, 162, 163 GIGMES, Didier......................................141, 200, 201 GILBERT, Helene....................................................323 GIMBERT, Yves......................................................107 GIRARD, Jean-Philippe.....................................65, 172 GIRARD, Victoria......................................................66 GIRARD, Vincent............................................251, 362 GIRAUD, Christophe..............................................261 GIROD, Marion...................................23, 61, 210, 362 GIULIANI, Alexandre.................53, 133, 293, 317, 362 GOFFINONT, Stphane..........................................271 GOGUET-SURMENIAN, Emilie................................204 GOLINELLI, Batrice...............................................139 GONDELLE, Hlne................................................245 GONNET, Florence.................................105, 115, 362 GONZALEZ-DE-PEREDO, Anne. . .23, 65, 109, 143, 172, 182, 183, 260, 319, 362 GOOSSENS, Pierre...................................................75 GOUHIER, Graldine..............................................257 GOULDEN, Pierre-Henry..........................................59 GRANIER, Claude...................................................311 GRASSEAU, Isabelle...............................................114 GRBAUT, Pascal...................................................195 GROSSEL, Martin...................................................152
374/381
GUDZINSKI, Ashley C..............................................120 GUELORGET, Amandine.........................................139 GUNIN, Erwann....................................................149 GUENNEAU-PUVIJESINGHE, Tania.........................266 GURINEAU, Vincent................................52, 139, 362 GUETTIER, Catherine.............................................284 GUIANVARC'H, Dominique....................................217 GUICHARD, Gilles...................................................171 GUIGLIARELLI, Bruno.............................................350 GUILHAUDIS, Laure................................................344 GUILIANI, Alexandre..............................................300 GUILLEN, Franck....................................................241 GUILLONNEAU, Franois....89, 95, 146, 160, 244, 287, 321, 362 GUILLOT, Alain.......................................151, 352, 362 GUILLOT, Laetitia...................................................239 GUITARD, Evelyne....................................................89 GUST, Marion........................................................326 GUTERL, Patrick.......................................................77
HOMER-VANNIASINKAM, Shervanthi....................126 HONDERMARCK, Hubert........................................150 HOPFGARTNER, Grard..................................108, 197 HOSTE, H...............................................................235 HOUNZANGBE-ADOT, M. S..................................235 HOURDEL, Vronique............................................157 HOVASSE, Agns............................................248, 363 HUART, Jean-Jacques.....................................233, 234 HUBERT-ROUX, Marie....................................257, 363 HUDSON, John.......................................................345 HUET, Sylvie...........................................................261 HUGHES, Chris.......................................................249 HUHMER, Andreas.................................................288 HUILLET, Cline........................................................99 HULMES, David........................................................69 HUMBEL, Stphane........................................141, 200 HRLIMANN, Hans C..........................................22, 74
-I-
-H-
HAAN, Serge..........................................................223 HACHANI, Johan....................................................203 HADDAD, Iman...............................................161, 310 HAFNAOUI , Norredine..........................................324 HAGGE, Agns..............................................211, 362 HAIECH, Jacques....................................................312 HASSAM JIJAKLI, M...............................................341 HAMDANE, Djemel................................................139 HAMM, Grgory.................................22, 73, 140, 340 HAMMANN, Philippe..............155, 171, 199, 272, 362 HAMON, Marion....................................................103 HAO, Zhiqi.............................................................288 HARDOUIN, Julie............................149, 153, 212, 362 HARICHAUX, Grgoire....................110, 114, 156, 362 HARSCOAT-SCHIAVO, Christelle.............................343 HARVENGHT, Luc...................................................335 HASHIMOTO, Masahiro.................................179, 298 HBERT, Yann........................................................111 HEBRAUD, Michel..........................................178, 262 HEEMSKERK, A. A...................................................273 HELARY, G..............................................................134 HLYE, Reynald......................................................198 HEM, Sonia.........................................25, 67, 308, 362 HNINGER, Michel.................................................330 HENION, Jack.................................................318, 322 HENRIQUES, Natali.........................................176, 362 HENRY, Cline................................129, 207, 352, 362 HERNIO, Nolwen....................................................239 HERSANT, Yael.......................................................105 HESSE, Anne-Marie........................................186, 362 HESTER, Alfons......................................................223 HEU, Cline....................................................325, 362 HEYMAN, Dominique.............................................228 HIRT, Heribert........................................................157 HIRTZ, Christophe..................................................181 HOLZMULLER, Philippe..........................................195
IBRAHIM, Marianne...............................................199 IDRISS, Hussein......................................................257 ITO, Yoshiyuki................................................179, 298
-JJACQUES FOURNIE, Jean........................................121 JAMIN, Emilien...................................26, 56, 123, 363 JAQUINOD, Michel............98, 102, 242, 267, 331, 363 JARDIN-MATH, Olivia...........................................157 JARNO, Nolwenn............................................331, 363 JARRAYA, Fayal....................................................311 JAULT, Jean-Michel................................................135 JEANNIN, Jean-Franois.........................................254 JEANVOINE, Yannick..............................................208 JGOU, Sandrine....................................................248 JOB, Claudette.......................................................251 JOB, Dominique.....................................................251 JOHANET, Catherine..............................................284 JOO CHUNG, Young...............................................256 JOUANIN , Isabelle.................................................123 JOUENNE, Thierry...........................149, 177, 212, 222 JOUMIER, Jean-Marc.............................................290 JOURDAN, Fabien....................................................56 JOURNET, Agns....................................................304 JOUVIN-MARCHE, E...............................................302 JOUX, Fabien.........................................................237 JUBEAUX, Guillaume..............................................214 JUILLARD, Vincent..................................................151 JULLIEN, Laure.......................................................107 JUNOT, Christophe.................................164, 181, 328
-K-
KAABIA , Zied.................................................122, 363 KARAKI, Samah........................................................48 KARAMANOS, Yannis.............................................203 KAROYAN, Philippe................................................217 KARSTEN, Marco....................................................285 KELLMANN, Markus...............................................288 KERBIRIOU, Mathieu..............................................338
375/381
KERDRAON, Olivier................................................276 KHARCHENKO, Andriy............................................223 KHRISTENKO, Nina.................................................288 KIEFFER-JAQUINOD, Sylvie.............102, 267, 304, 331 KIEHNE, Andrea.....................................................111 KIFAGI, Chamseddine............................................311 KING, Jay D............................................................222 KIRSCH, Gilbert......................................................180 KISS, Agnta.......................................................26, 82 KLEIN, Grard........................................................304 KLEIN, Tania...........................................................352 KRAUT, Alexandra..............................8, 186, 267, 363 KRISTIAN HANNIBAL-BACH, Hans..........................281 KUBO, Ayumi.................................................179, 298 KUHN, Lauriane........................91, 155, 199, 272, 363 KULESZA, Alexander..............................................184 KULYK, Hanna........................................................123 KUSAI, Akihiko..................................19, 179, 298, 363 KUZMINSKA, Maryna.............................................351 KWASIBORSKI, Anthony.................................341, 363
-LL'HOSTIS, Guillaume..............................................164 LABADIE-LEMIRE, Emilie......................................253 LABAS, Valrie................110, 113, 114, 156, 189, 363 LABEL, Philippe......................................................335 LACOMBE, Claire..............................................68, 187 LACOMBE, Jrme...........................................96, 363 LACOUX, Xavier.......................................................66 LACROIX, Chrystelle.......................................260, 363 LADJIMI, Moncef.....................................................93 LAFAY, Florent.......................................................334 LAFITTE, Daniel...........................22, 78, 282, 309, 363 LAFOSSE, Michel....................................................142 LAGARRIGUE, Mlanie...................................232, 238 LALMANACH, Anne-Christine.................................110 LAMARCA, Arnaud.........................................295, 363 LAMBERT, Jean Charles..........................................138 LAMBERT, Marine..........................................119, 363 LAMOURETTE, Patricia.............................................75 LAMRANI, Myriam.................................................254 LANE, Cathy...........................................................234 LANGE, Catherine...........................152, 257, 353, 363 LANGELLA, Olivier............................................83, 263 LANGRIDGE, James................................................348 LANO, Laura.........................................................182 LAPAILLERIE, Delphine...........................................188 LAPRVOTE, Olivier. .53, 108, 131, 140, 159, 185, 193, 240, 245, 340, 363 LARDENOIS , Aurlie................................................72 LARR, Colette.......................................................103 LARTIA, Remi.........................................................107 LARVOR, Frederic...................................................221 LATAILLADE, J.J......................................................134 LATCHE, Alain........................................................336 LAUMAIN, Bastien...................................................95 LAUNAY, Jean-Claude............................................186
LAURENT, Victor....................................................250 LAURIER, Marie......................................................262 LAVANANT, Hlne........................................165, 363 LAVIGNE, Rgis...................22, 72, 230, 232, 239, 363 LAZAR, Adina N,.....................................................240 LE BIZEC, Bruno.............9, 57, 122, 137, 221, 247, 363 LE BOURHIS, Xuefen..............................................150 LE CAER, Jean-Pierre..............................................284 LE GALL, Caroline...................................................260 LE GALL, Morgane............................................95, 146 LE GORREC, Madalen...............................................85 LE MARECHAL, Pierre.....................................166, 363 LE QUR, Jean-Luc........................................168, 175 LE VOT, Clotilde.....................................................330 LEBARON, Philippe.................................................237 LEBEAU, Diane...............................................124, 363 LEBERT, Dorothe....................................................99 LEBLANC, Catherine...............................................124 LEBLOIS, Thrse...................................................307 LEBOULENGER, Franois........................................274 LEBRET, Bndicte.................................................323 LEBRETON, Mlanie.........................................91, 198 LEBRUN, Rgine.............................................350, 363 LECAMP, L..............................................................353 LEDUC, Marjorie...............................95, 244, 321, 364 LEFAY, Catherine...................................................201 LEFRANC, Marie-Paule.............................................89 LEGOUFFE, Raphael..................................73, 140, 340 LEGROS, Vronique...............................................300 LEHMANN, Sylvain.................................................181 LEIZE-WAGNER, Emmanuelle.................272, 273, 347 LEIZE, Emmanuelle....91, 198, 199, 272, 273, 347, 364 LELU-WALTER, Marie-Anne...................................335 LEMAIRE, Jol........................................................330 LEMOINE, Jrme......54, 66, 138, 143, 183, 191, 192, 210, 214, 280, 364 LENNON, Sarah......................................278, 312, 364 LONARD, Didier...................................................333 LEPOIVRE, Philippe................................................341 LEPRINCE, Jrme..................................................344 LERICHE, Emma-Dune....................................152, 364 LEROY, Eric.............................................235, 265, 364 LEROY, V................................................................302 LESAGE, Denis........................................303, 320, 364 LESCURE, Marie-Hlne.........................................269 LESSIM, S...............................................................134 LESSIRE, Ren........................................................106 LETENDRE, Julie.....................................................274 LI-BEISSON, Yonghua.............................................101 LIBAT, Stphane.....................................................252 LIBONG, Danielle...........................................320, 364 LIGNON, Sabrina............................................350, 364 LILLA, Sergio..........................................................163 LISACEK, Frederique................................................71 LIU , Yuchen.....................................................72, 230 LOBINSKI, Ryszard..................................194, 290, 329
376/381
LOFTUS, Neil..................................................196, 197 LOISEAU , Florence..................................................97 LOKIEC, Francois....................................................118 LONGUESPE, Rmi.......................................276, 364 LNNBERG, Maria...................................................81 LOPPINET-SERANI, Anne........................................269 LORENZI, Magali....................................................350 LOUIS BEAUDEUX, Jean.........................................108 LOUTELIER-BOURHIS, Corinne........152, 165, 353, 364 LOUWAGIE, Mathilde.....................................102, 364 LUCCHI, Graldine..........................254, 255, 307, 325 LUCIO, Marianna.....................................................82 LUTOMSKI, Didier..........................................134, 228 LYAN, Bernard................................................270, 364
-MMACALEESE, Luke..................................................191 MAGALLON, Thierry...............................................110 MAINGONNAT, Jean-Franois................................207 MAJERAN, Wojciech......................................162, 163 MAJOR, Mohamed.........................................201, 364 MAJOR, Yannis...............................................202, 364 MAKAROV, Alexander............................................116 MALARD, Vronique..................................8, 283, 364 MALATS, Nria.........................................................85 MALOSSE, Christian..........................84, 120, 132, 364 MAN, Petr..............................................................135 MANACH, Claudine................................................270 MANG, Alain..................................................96, 364 MANN, Carl............................................................167 MANNOURY LA COUR, Clotilde..........................48, 97 MARC, Ivan............................................................343 MARCELLIN, Didier.........................................314, 364 MARCELLIN, Marlne.............................277, 282, 364 MARCHAND, Philippe............................................221 MARCHE, Patrice N. ......................................295, 302 MARCHETTI, Catherine..........................................245 MARI, Adriano.......................................................161 MARIE LOMENECH, Anne......................................188 MARIE, Cecile........................................................349 MARIN, Philippe...............................9, 48, 76, 97, 364 MARQUE, Sylvain...................................................350 MARTEAU, Charlotte.............................................137 MARTELET, Armelle.......................................164, 364 MARTELLI, Alain.......................................................92 MARTIGNAC, Marion.....................................147, 364 MARTIN, Benot.....................................................232 MARTIN, Nicolas....................................................255 MARTIN, Patrice.............................................352, 364 MARTINEZ, Aude...................................................163 MARTINEZ, Jean.................................55, 64, 136, 173 MARTINHO, Marlne.............................................350 MARTROU, David...................................................169 MARTY-GASSET, Nathalie...............................218, 364 MARULLO, Philippe................................................261 MARVIN-GUY, Laure F...........................................256 MARY, Jean....................................................339, 364
MASSELON, Christophe D...........24, 85, 236, 304, 364 MASSON, Gilles......................................................119 MATALLANA-SURGET, Sabine................................237 MATHERON, Lucrce.....................................217, 364 MATONDO, Mariette.....................................277, 364 MAUCLAIRE, Grard..............................................330 MAUCOURT, Mickal.............................................253 MAURETTE, Mat.................................................147 MAYBORODA, O.A.................................................273 MAYEUX, Patrick.................89, 95, 244, 287, 321, 364 MAZA, Elie.............................................................336 MAZEROLLES, Grard............................................119 MECHKARSKA, Milena...........................................222 MCHULAM, Yves..........................................132, 297 MEETANI, Mohammed A.......................................222 MEFFRE, Julie...........................................................97 MEHMOOD, Shahid...............................................135 MEINNEL, Thierry.............................63, 162, 163, 364 MEIRELES, Maria-Hlna.........................................56 MEJEAN, Arnaud....................................................342 MEKLAT, Noura..............................................270, 364 MELKI, Ronald........................................................249 MELLAL, Mourad..............................................85, 236 MEMBOEUF, Antony..............................................107 MENAND, Benot...........................................104, 365 MENCH, Michel.....................................................269 MENEI, Philippe.....................................................238 MERCADIER, Jean-Jacques.....................................146 MESMIN, Cdric.....................................................301 MESTDAGH, Hlne...............................................330 MEUDEC, Emmanuelle...................................119, 365 MEUNIER-GONTERO, Brigitte................................350 MICHAEL CONLON, J..............................................222 MICHALAK, Sophie.................................................238 MICHEL, Thomas....................................................125 MICHELLAND, Sylvie..............................................220 MICHOPOULOS, Filippos........................................196 MIGNEN, Olivier....................................................338 MIGONNEY, V........................................................134 MIGOT-NABIAS, Florence.........................89, 321, 365 MIGUIRDITCHIAN, Manuel....................................349 MIJAKOVIC, Ivan....................................................129 MILEO, Elisa...........................................................350 MILLAN, Mark J..................................................48, 97 MILOHANIC, Eliane................................................129 MILOSAVLJEVIC, Aleksandar..................................317 MINSINI, Carole.....................................................234 MIRANDA, Guy...............................................352, 365 MISRA, Sandeep....................................................129 MITRIC, Roland......................................................184 MOALI, Catherine.......................................23, 69, 365 MOEHRING, Thomas......................................116, 288 MOING, Annick......................................................253 MOLETTE, Caroline................................................218 MOLINA, Franck.....................................................311 MOLINA, Laurence.........................................311, 365
377/381
MONGRAND, Sbastien...........................................51 MONNET, Vronique.....................................129, 151 MONNEUSE, Jean-Marc...................................67, 365 MONNIER, Valrie..................................141, 200, 365 MONSARRAT, Bernard.......60, 65, 109, 143, 172, 182, 183, 246, 250, 252, 259, 260, 277, 282, 289, 319, 365 MONTAN, Marie-Hlne..............................104, 365 MONTAVON, Philippe............................................256 MONTEAU, Fabrice..................................................57 MOREAU, Stphane.........................19, 196, 197, 365 MOREIRA, Guillaume.............................................201 MOREL, Alexandre.................................................335 MOREL, Natalie........................................................75 MOREL, Paul..................................................278, 365 MORIKAWA, Yoshio...............................................196 MORLET, Lise.........................................................191 MORLOCK, Gerda..................................................354 MOUCHE, Claire.............................................268, 365 MOULIN, Morgane................................................245 MOULS, Laetitia.............................................112, 365 MOUTON-BARBOSA, Emmanuelle...........65, 109, 172 MOUZ, Nicolas.........................................................99 MOYET, Lucas........................................................236 MOZZO, Milena.....................................................104 MUELLER, Mathias.................................................116 MULLER, Bruno......................................................164 MULLER, Catherine................................................250 MULLER, Julie........................................................349 MULLER, Ludovic...................................................225 MULLER, Markus........................................26, 71, 365
OLIVEROS, Esther...................................................147 OLIVIER, Audrey.....................................................259 OLOUNLAD, P. A..................................................235 ONODERA, Jyun.....................................................298 OPENSHAW, Matthew E................................128, 309 ORIVEL, Jrme.....................................................344 ORTIZ, Alexia..........................................233, 234, 365 ORTIZ, Daniel.........................................................213 OSORIO-ALGAR, Sonia...........................................253 OUARDAD, Samira.................................................268 OUERDANE, Laurent...............................194, 329, 365 OUGHLIS , S...........................................................134 OUIDIR, Tassadit....................................................212 OVERALL, Christopher..........................23, 42, 69, 365
-PPAGE, Adeline..................................................92, 365 PAGS, Frderic.....................................................227 PAIN-DEVIN , Sandrine...........................................177 PAMELARD, Fabien..................................................73 PANCHAL, Ma...............................................185, 240 PANCHOUT, Franois.............................................274 PANSANEL, Jrme..................................................77 PANVERT, Michel...................................................132 PARANT , Marc......................................................177 PARIS, Alain...........................................................328 PARIS, Cdric.........................................................343 PARRA GIMENEZ, Nereida.....................................195 PASCHKE, Carmen..................................................116 PASCUAL, Aurlie...................................................227 PECH, Jean-Claude.................................................336 PCHIN, Sverine.................................................174 PLEGRIN, Andr.....................................................96 PELOSI, Ludovic.....................................................135 PELTIER, Gilles.......................................................224 PELTIER, Julien.......................................................229 PEPE, Claude..........................................................303 PROT-TAILLANDIER, Marie.....................80, 280, 365 PERROT, Michel......................................127, 188, 365 PERUCH, Frdric..................................................268 PETER SCHANSTRA, Joost......................................260 PETIOT, Stphanie..........................................278, 292 PETIT, Vanessa.......................................................240 PFLIEGER, Delphine........................................157, 365 PHAN, Trang N.T....................................141, 200, 202 PIBLE, Olivier..............................................8, 315, 365 PICARD, Guillaume.............................................98, 99 PICAUD, Vincent....................................................130 PICHEREAUX, Carole.......................218, 252, 336, 365 PIERRE, Jonlet........................................................216 PIGUET, Dominique...............................................256 PINARD, Amlie.....................................................352 PINEAU, Charles............9, 72, 230, 232, 238, 239, 365 PINGUET, Jrmy...........................................324, 366 PINSON, Benot................................................74, 366 PIVETEAU, Catherine...............................................73 PIZZALA, Hlne....................................................202
-NNAHON, Laurent....................................133, 293, 317 NAMANE, Abdelkader............................................145 NAMY, Olivier........................................................158 NANO, Edlira..................................................263, 365 NASSO, Sara...........................................................289 NAUBOURG, Pierre................................................209 NAUDIN, Bertrand.........................................177, 365 NDIAYE, Sega.................................................296, 310 NEGRONI, Luc................................................243, 365 NESPOULOUS, Claude............................................308 NETTER, Claude................................19, 285, 286, 365 NGUYEN-KHOA, Thao............................................342 NGUYEN, Mai.........................................................101 NGUYEN, Uyen...............................................279, 337 NGUYEN, Viet........................................................225 NICOD, Laurence...................................................325 NICOL, Edith...............................24, 58, 148, 297, 365 NICOLI, Raul...........................................................220 NIETO, Laurence....................................................319 NIKITIN, Frederic......................................................71 NITSCH, Robert......................................................281
-O-
OHARA, Keiichiro.....................................................55 OJO, Opeolu O.......................................................222
378/381
PLANCHON, Stella..................................................207 PLANELLES, Gabrielle.............................................342 PLECHAKOVA, Olga................................................258 PLOU , Philippe......................................................122 POCK, Katharina....................................................128 POINOT, Pauline....................................................181 POINSOT, Vrna.....................................................90 POIRI, Marylne...................................................204 POIRIER, Florence...................................134, 228, 366 POLARD, Patrice....................................................259 POLLET, Samuel.....................................................147 PONCET, Pascal..............................................161, 366 PONT, Frdric...............................................121, 366 POPHILLAT, Matthieu....................................100, 366 POPOT, Marie-Agns.......................................81, 328 PORBECK, Frank.....................................................299 PORTAIS, Jean Charles.............................................90 POTIER, Noelle.........................................91, 198, 366 POUECH, Charlne.........................................333, 334 POURCHER, Thierry........................................124, 314 POURHASSAN, Nina.......................................273, 347 POUSSEREAU , Nathalie.........................................251 PREUD'HOMME, Hugues................................194, 290 PREVILON, Miresta................................................146 PRIMIG , Michael.............................................72, 230 PROSSER, Simon............................................318, 322 PRULL-JANSSEN, Mehdi.........................................105 PRZYBYLSKI, Cdric.................................105, 115, 366 PUCCIO, Hlne.......................................................92 PUECH PAGS, Virginie............................................90 PUJOS-GUILLOT, Estelle..........................270, 324, 366 PUPPO, Carine.......................................................350
-QQUANICO, Jusal..............................................351, 366 QUAU, Herv.......................................................214 QUINTANA, Mercedes...........................................270
-R-
RENAUT, Jenny......................................................341 RENIER, Sandra..............................................178, 366 REUTENAUER, Laurence..........................................92 REY, Martial...........................................................135 REYNAUD, Karine...................................................156 REZAI, Keyvan........................................................118 RHOURRI-FRIH, Boutayna......................................142 RICHA, Latifa..........................................................234 RIFFLET, Aline........................................................344 ROBAGLIA, Christophe...........................................104 ROBBE, Marie-France.............................130, 223, 366 ROBINE, Ophlie............................................148, 366 ROCHE, Serge........................................................246 ROCHER, Batrice..................................................274 ROEMPP, Andreas.................................................223 ROFIDAL, Valrie............................................308, 366 ROGNIAUX, Hlne........................................103, 366 ROLANDO, Christian....24, 44, 215, 233, 234, 258, 366 ROLANDO, Monica.................................................145 ROLIN, Dominique.................................................253 ROLLAND, Norbert.................................224, 236, 305 ROMIEU, Anthony..................................................120 RONAT-HEIT, Evelyne............................................343 ROPARTZ, David.............................................103, 366 ROPERCH, Jean-Pierre...........................................229 ROSENBAUM, Jean................................................243 ROSSIGNOL, Michel..........................67, 218, 308, 336 ROUET-BENZINEB, Patricia.....................................146 ROULEAU, Alain.....................................................307 ROUSSET, Jean-Pierre............................................158 ROUSSI, Stamatiki..................................................283 ROUX-DALVAI, Florence.................109, 143, 183, 366 ROYLE, Louise........................................................128 ROZAND, Christine.................................................164 RUBIANO-LABRADOR, Carolina..............................291 RUCH, David............................................................70 RUDAZ, Serge........................................................220
RABILLOUD, Thierry.................................................88 RAMPAL, Sushma..................................................346 RAMZAN, M...........................................................302 RASCLE , Christine..................................................251 RAVALLEC, Marc....................................................204 RAYNAL, Patrick.....................................................246 REBUF, Christian....................................................204 REBUFFAT, Sylvie...........................................190, 280 REDEKER, Virginie..........................................249, 366 REDON, Sebastien....................................................54 REFREGIERS, Matthieu...........................................293 RGAZETTI, Anne...................................................193 REILLE-TAILLEFERT, Genevive..............................258 REILLER, Pascal......................................................124 REMOUE, Franck....................................................227 REMY-MARTIN, Fabien..........................................307 RENARD, Pierre-Yves.............................................120 RENAUD, Jean-Paul................................................292
-S-
SABLIER, Michel.......................................................59 SACHON, Emmanuelle................25, 68, 187, 217, 366 SAGAN, Sandrine...........................................206, 366 SAGE, Nicole..............................................8, 283, 366 SAGO, Lala.......................................89, 287, 321, 366 SAINT-DIZIER, Marie..............................................156 SAKR, Samer..........................................................166 SALIOU, Jean-Michel..............................................312 SALNOT, Virginie.........89, 95, 160, 244, 287, 321, 366 SALPIN, Jean-Yves..........................................208, 213 SALVADOR, Arnaud....................54, 66, 138, 214, 366 SALVETAT, Nicolas.........................................311, 366 SALVI, Daniel..................................................236, 305 SALZET, Michel...................................9, 276, 351, 366 SAMUEL, Didier.....................................................284 SANGLIER-CIANFRANI, Sarah........................292, 312 SANTONI, Vronique...............................................67 SANTORO, Massimo..............................................275
379/381
SARNI-MANCHADO, Pascale..................................317 SATO, Takafumi.....................................................179 SATOH, Takaya......................................................179 SAVISTCHENKO, Jimmy..........................................249 SAVOUREY, Gustave..............................................186 SAYD, Thierry.........................................323, 324, 366 SCHAEFFER-REISS, Christine...........................248, 366 SCHEFFLER, Jean-Luc...............................................62 SCHEMBRI, Therese.................................78, 309, 366 SCHILTZ, Odile................................................252, 366 SCHMIT, Pierre-Olivier...................................111, 272 SCHMITT-KOPPLIN , Philippe...................................82 SCHMITT, Emmanuelle...................................132, 297 SCHMITTER, Jean-Marie...................................51, 243 SCHMUCKER, Stphane...........................................92 SCHOENMAKER, B.................................................273 SCHRAMM, Sbastien................................24, 62, 366 SCHULZ-UTERMOEHL, Timothy..............................196 SCHWIKOWSKI, Benno...........................................157 SEBAGH, Mylne....................................................284 SEIGNEURIN-BERNY, Daphn.................................236 SEIMANDI, Mathieu.................................................76 SELLAMI, Lyna................................................309, 367 SMON, Etienne.....................................168, 175, 367 SENECHAL, Hlne.........................................161, 367 SERGEANT, Nicolas................................................327 SEVE, Michel............................................71, 220, 367 SVENO, Martial..............................................97, 195 SEYER, Alexandre...........................................144, 367 SEYER, Pascal.............................................23, 76, 367 SHAHALI, Youcef............................................161, 367 SHEN, Shida...........................................................275 SIBILLE, Estelle...............................................180, 367 SICARD, Delphine...................................................261 SILVA ZOLLEZZI, Irma.............................................256 SIMON, Romain................................54, 138, 214, 367 SMARGIASSO, Nicolas............................................216 SMIETANA, Michael.................................................55 SNEEKES, Evert-Jan........................................285, 286 SOBESKY, Rodolphe...............................................284 SOBHANI, Iradj.......................................................229 SOLASSOL, Jrme...................................................96 SOMAIYA, Pranav..................................................126 SOMMERER, Nicolas..............................................119 SPECHT, Michael....................................................224 SPENCER, Daniel....................................................128 SPEZIA, Riccardo....................................................208 SPINA, Lucie...........................................................113 STAUBER, Jonathan..........................73, 140, 340, 367 STEINMETZ, Michel O............................................282 STELLA, Alexandre..........................143, 183, 259, 367 STEUNOU, Anne-Lise.............................................319 STOECKLI, Markus..................................................223 STOJILJKOVIC, Natali..............................................328 STBIGER, Gerald..................................................264 STURM, N..............................................................302
SUGANO, Madeleine...............................................67 SUMPTON, David...................................................163 SUTRA, Jean-Pierre................................................161 SWART, Remco..............................................285, 286
-TTABET, Jean-Claude..80, 144, 164, 190, 205, 225, 280, 303, 328 TAIYA, Mehdi.........................................................338 TAJAN, Mylne......................................................246 TAM HA, T. B..........................................................235 TAMURA, Jun.................................................179, 298 TANGUY, Arnaud...................................................339 TARDIF, Marianne..........................................224, 367 TEIXEIRA-GOMES, Ana-Paula. .110, 113, 114, 189, 239 TENE, Nathan.................................................344, 367 TENG, Ling.............................................................338 TERLOT, Jean-Didier...............................................114 TERRAS , Lionel......................................................282 TEYSSIER, Caroline.................................................335 THERETZ, Alain.......................................................164 THRON, Laetitia............................................170, 218 THVENOT, Etienne...............................................223 THILLAYE DU BOULLAY, Olivier..............................265 TIERS, Laurent........................................................181 TITMAN Chris.................................................196, 197 TITMAN, Chris................................................196, 197 TOILLON, Robert-Alain...........................................150 TOKARSKI, Caroline................................233, 234, 258 TOLEDANO, Michel................................................296 TONG, Germain...............................................68, 187 TORIBIO, Alix.........................................................125 TOUBOUL, David....23, 52, 53, 94, 131, 139, 170, 185, 240, 367 TOURNIER, Mikal.................................................334 TOWERS, Mark......................................................348 TRAUCHESSEC, Mathieu.........................242, 278, 367 TRGUER, Karine...................................................246 TREILHOU, Michel..................................................344 TREMBLAY-FRANCO, Marie.....................................56 TRIBOULET, Sarah............................................88, 367 TRIMAILLE, Thomas.......................................141, 200 TRONTIN, Jean-Franois........................................335 TROUVE, Pascal.....................................................338 TRUNTZER, Caroline...............................................209 TSA, Chia-feng.......................................................111 TSIBYN, Yury..........................................................192 TSIKIS, Guillaume...................................................189 TUTUNDJIAN, Renaud............................................214
-U-
UBUKATA, Masaaki........................................179, 298 UTTENWEILER-JOSEPH, Sandrine...........................277 UZBEKOVA, Svetlana......................................113, 367
-VVALENTIN, A..........................................................235 VALET, Philippe......................................................250
380/381
VALLE, Cyrille...............................................221, 367 VALLON, Olivier.....................................................224 VALOT, Benot.....................26, 83, 207, 261, 263, 367 VAN AGTHOVEN, Maria A......................................215 VAN DER REST, Guillaume58, 120, 132, 148, 297, 317, 367 VAN DORSSELAER, Alain....77, 88, 248, 292, 294, 312, 367 VAN LING, Robert..........................................285, 286 VANDENBOGAERT, Mathias...................................157 VANDENBROUCK, Yves......................................8, 304 VANDERMOERE, Franck.......................25, 48, 76, 367 VARESIO, Emmanuel......................................108, 197 VASSEUR, Jean-Jacques...........................................55 VERBAERE, Arnaud................................................119 VERDIER-PINARD, Pascal........................................282 VERDIER, Yann.......................................310, 327, 367 VERGELY, Isabelle..................................................209 VETILLARD, Anglique............................................344 VEUTHEY, Jean-Luc................................................220 VIALA, Cline.........................................................174 VIALA, Didier..................................218, 262, 324, 367 VIANELLO, Sara..................................................52, 94 VIDAUD, Claude.............................................211, 367 VIEL, Stphane...............................................141, 200 VIEU, Diane-Lore....................................................342 VILAIN, Sbastien...........................127, 188, 226, 367 VILLARD, Claude...............................78, 282, 309, 367 VILLAUME, Sandra.................................................248 VILLIERS, Christian L...............................................295 VILLIERS, Florent....................................................331 VINATIER, Denis.....................................................276 VINH, Jolle.............117, 161, 296, 310, 327, 339, 367 VISSERS, Johannes P.C...........................................249 VITRY, Christiane............................................268, 367 VIVAT HANNAH, Valrie.........................................292 VLAK, Kees...............................................19, 281, 367 VOGT, Johannes.....................................................281
WERNER, Erwan....................................................185 WERTHER, Wolfgang.............................................264 WEST, Caroline......................................................142 WEY, Emmanuel....................................................126 WIEST, Laure..........................................326, 333, 334 WILLAND, Nicolas....................................................73 WILLIAM DUPUY, Jean...........................................188 WILSON, Ian D.......................................................196 WIRTH, Jrmie.....................................................119 WISZTORSKI, Maxence...................140, 231, 340, 351 WOLFF, Philippe.............................................171, 368 WU, Jianqing..................................................297, 368
-XXU, Hao..................................................................261 XU, Ying.................................................................225 XUE, Baiyie.............................................................225
-Y-
YANG, Yang..............................................................50 YART, Armelle........................................................246 YERLY, Justine........................................................256 YSABEL GONZATTI, Mary.......................................195 YU BAO, Cong........................................................154
-ZZACCARIA, Affif..............................................313, 368 ZAL, Franck............................................................198 ZALKO, Daniel..................................................56, 137 ZALTSMAN, Yefim..................................................206 ZAMBARDI, Gilles.....................................................66 ZANELLA-CLON, Isabelle........................................69 ZARSKI, J-P.............................................................302 ZEGGARI, Rabah....................................................307 ZELLER, Martin.......................................................116 ZENDONG, Zita.......................................221, 247, 368 ZENOBI, Renato.......................................................50 ZEYER, Denis..........................................................292 ZGANI, Ibrahim......................................................257 ZHANG, Wenjing....................................................154 ZHAO, Jingjing........................................................120 ZIARELLI, Fabio......................................................202 ZINS, Emilie-Laure..................................................303 ZIRAH, Sverine.............................................190, 280 ZISKIND, Michael...................................................231 ZIVY, Michel.............................................83, 261, 263 ZORZ, Nicole..........................................................349 ZOUINE, Mohamed................................................336 ZOVI, O..................................................................353
-WWAGNER, Michel...................................................197 WAGNER, Renaud....................................................91 WANG, Fen..............................................................51 WASSELIN, Thierry.................................................294 WATTENHOFER-DONZ, Marie................................92 WATTIEZ, Ruddy....................................................237 WAXWEILER, Sophie..............................................216 WEILL, Sophie........................................................118
381/381
Congrs gr conjointement par : la SFEAP & la SFSM Laboratoires organisateurs : CEA/iBEB/SBTN/LBSP CEA/iRTSV/BGE/EDyP

Abstract Book All

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Abstract Book All

Uploaded by

Copyright:

Available Formats

SMAP Conference, September 19th-22nd 2011, Avignon

Popes' Palace photos credit : Yann Fareins / Noir d'Ivoire

SMAP Conference, September 19th-22nd 2011, Avignon

Participants list.........................................................................357 Author Index.............................................................................369

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

Philippe Dugourd, Laboratoire de Spectromtrie Ionique et Molculaire, UMR5579

Bruno le Bizec, LABERCA - U1329 INRA, Ecole Nationale Vtrinaire, Agroalimentaire et de

Michel Salzet, Laboratoire de Spectromtrie de Masse Biologique Fondamentale et Applique

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

Popes' palace plan

SMAP Conference, September 19th-22nd 2011, Avignon

1-Salles des Gardes : Welcome /

3-Paneterie : Poster sessions n1 & n2

5-Conclave : Plenary conferences &Oral

7-Chambre du trsorier : Manufacturer

12-Grande audience : Exhibitors room &

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

Oral sessions organisation

Instructions for oral communication

Organisation des confrences

Instructions pour les confrences slectionnes

Dure des prsentations : 15 min prcisment et 5 min gres par le modrateur

SMAP Conference, September 19th-22nd 2011, Avignon

Poster sessions organisation

Organisation sessions posters

SMAP Conference, September 19th-22nd 2011, Avignon

Mr Sergio MELIS Mr Guillaume CHARLOT

21 - LGS LAB GAZ SYSTEMS

4 - ASA - ADVANCED SOLUTIONS ACCELERATOR

Mr Gerard VAN DER LAAN Mr Nico WORTEL

Mr Patrick BELLEMIN Mr Stphane FESSEMAZ

Mrs Karima BAUDIN Mr Christophe CLARYSSE

8 - PROTEINSIMPLE (CELL BIOSCIENCES)

27 - PROTEOME SOFTWARE 28 - SERVA ELECTROPHORESIS GMBH

Mr Pierre BERNARD SAVARY

11 - CLUZEAU INFO LABO

Mr Mikael LEVI Mr Stphane MOREAU

30 - TECAN FRANCE SAS

31 - THERMO ELECTRON SAS

Mr Jean- Louis SCHAFFAR Mr Gatan SEEBURN

Mrs Fabienne PALGE

Mrs Sophie BERTAUX

Mr Akihiko KUSAI Mr Jean- Pierre MUNIER

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

SMAP Conference, September 19th-22nd 2011, Avignon

Monday September 19th

Conclave (5) 5:30pm-6:30pm

SMAP Conference, September 19th-22nd 2011, Avignon

Tuesday September 20th

9:35am-12:30am 9:35am 10:10am 10:30am Conclave (5)

Paneterie (3) Grde Audience (12)

10:30am Paneterie (3) Grde Audience (12) 11:30am

12:10 12:40am-1:55pm 12:55am-2:25pm 1:40pm-2:25pm 2:00pm-2:25pm

SMAP Conference, September 19th-22nd 2011, Avignon