Proteo Mics

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Proteomics/Printversion
Proteomics
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IntroductiontoProteomics
Presentation
Printversion
Contents
1 IntroductiontoProteomics
1.1 Presentation
1.2 Whatisproteomics?
1.3 Theimportanceofproteomics
1.4 ProteomicsWorkflows
1.5 BroadBasedProteomics
1.6 References
1.7 ArticlesSummarized
1.7.1 AdvancesinProteomicWorkflowsforSystemsBiology
1.7.2 BroadBasedProteomicStrategies:APracticalGuidetoProteomicsandFunctionalScreening
1.8 WebsitesSummarized
1.8.1 TheAssociationofBimolecularResourceFacilities:ProteomicsResearchGroup(PRG)
1.8.2 IntroductiontoProteomics
1.8.3 IntroductiontoProteomics
1.9 References
2 ProteinSamplePreparation
2.1 Presentation
2.2 Introduction
2.3 Bibliography
3 PlantProteomicsaboutTwoDimensionalGelElectrophoresis
https://en.wikibooks.org/wiki/Proteomics/Print_version 1/38
3.1 Presentation
4 ProteinSeparationsChromatography
4.1 Presentation
4.2 Introduction
4.3 Resources
4.4 References
5 ProteinSeparationsElectrophoresis/IntroductiontoElectrophoresis
5.1 Presentation
5.2 Definitions
5.3 ElectrophoresisTheory
5.4 ApplicationsofElectrophoresis
5.5 References
5.6 MicrofluidicElectrophoresis
5.6.1 WhatWillProteomicsGainfromMicrofluidic?
5.6.2 ElectrophoresisinMicrofluidicMicrosystems
5.6.3 MicrofluidicGelElectrophoresis
5.6.3.1 I.FabricationProcess
5.6.3.2 II.PrincipleofOperation
5.6.3.3 III.ClinicalApplications
5.6.4 MicrofluidicCapillaryElectrophoresis
5.6.4.1 I.FabricationProcess
5.6.4.2 II.PrincipleofOperation
5.6.4.3 III.ClinicalApplications
5.6.5 Notes
5.6.6 References
6 ProteinSeparationsCentrifugation
6.1 Presentation
6.2 IntroductiontoCentrifugation
6.3 UsesofCentrifugation
6.4 References
6.4.1 SubscriptionBasedReferences
6.4.2 OpenAccessReferences
7 EmergingandMiscellaneousProteomicsTechnologies
7.1 Presentation
8 EmergingandMiscellaneousTechnologiesinProteomics
8.1 XRayTomography
8.2 IntroductiontoProteoinformatics
8.2.1 ProteinIdentificationDatabase
8.2.1.1 WhatareProteinIdentificationDatabases?
8.2.1.2 TheFutureofProteinIdentificationDatabases
8.2.2 NewTechniquesinImageAnalysis
8.3 LaserCaptureMicrodissection
8.4 ProteomicComplexDetectionusingSedimentation
8.5 References
9 ProteinIdentificationMassSpectrometry
9.1 Presentation
9.2 Introduction
9.3 References
10 ProteinPrimaryStructure
10.1 Presentation
10.2 Introduction
11 PosttranslationalModification
11.1 Presentation
11.2 Introduction
12 ProteinProteinInteractions
12.1 Presentation
12.2 Introduction
13 ProteinChips
13.1 Presentation
13.2 Introduction
13.3 History
13.3.1 NucleicAcidMicroarrays
13.3.2 DeficiencyofTraditionalProteinCharacterizationMethods
13.3.3 ProteinChipPrecursorstoModernDay
13.4 References
14 ProteomicsandDrugDiscovery
14.1 Presentation
14.2 IntroductiontotheDrugDiscoveryProcess
14.2.1 References:
15 Biomarkers
15.1 Presentation
15.2 Massspectrometrybasedtargetedproteinquantification:methodsandapplications
15.3 MainFocus
15.4 Newterms
15.5 Summary
15.6 RelevancetoProteomicsCourse
15.7 References
16 ExperimentalProtocols
16.1 Presentation
17 Contributors
18 Contributorslist
Whatisproteomics?
Thefocusofproteomicsisabiologicalgroupcalledtheproteome.Theproteomeisdynamic,
definedasthesetofproteinsexpressedinaspecificcell,givenaparticularsetofconditions.
Withinagivenhumanproteome,thenumberofproteinscanbeaslargeas2million.[1]
Proteinsthemselvesaremacromolecules:longchainsofaminoacids.Thisaminoacidchainis
constructedwhenthecellularmachineryoftheribosometranslatesRNAtranscriptsfromDNAin
thecell'snucleus.[2]Thetransferofinformationwithincellscommonlyfollowsthispath,from
DNAtoRNAtoprotein.
Proteinscanbeorganizedinfourstructurallevels:
Primary(1):Theaminoacidsequence,containingmembersofa(usually)twenty
unitalphabet
Secondary(2):Localfoldingoftheaminoacidsequenceintohelicesandsheets
Tertiary(3):3Dconformationoftheentireaminoacidsequence
Quaternary(4):Interactionbetweenmultiplesmallpeptidesorproteinsubunitsto Informationtransferinthecentral
createalargeunit
dogmaofbiology
Eachlevelofproteinstructureisessentialtothefinishedmolecule'sfunction.Theprimary
sequenceoftheaminoacidchaindetermineswheresecondarystructureswillform,aswellastheoverallshapeofthefinal3D
conformation.The3Dconformationofeachsmallpeptideorsubunitdeterminesthefinalstructureandfunctionofaprotein
conglomerate.[3]
Therearemanydifferentsubdivisionsofproteomics,including:
Structuralproteomicsindepthanalysisofproteinstructure
Expressionproteomicsanalysisofexpressionanddifferentialexpressionofproteins
Interactionproteomicsanalysisofinteractionsbetweenproteinstocharacterizecomplexesanddeterminefunction.[4]
Proteomicshasbothaphysicallaboratorycomponentandacomputationalcomponent.Thesetwopartsareoftenlinkedtogetherattimes
dataderivedfromlaboratoryworkcanbefeddirectlyintosequenceandstructurepredictionalgorithms.Massspectrometryofmultiple
typesisusedmostfrequentlyforthispurpose.[5]
Theimportanceofproteomics
Proteomicsisarelativelyrecentfieldthetermwascoinedin1994,andthescienceitselfhaditsoriginsinelectrophoreticseparation
techniquesofthe1970'sand1980's.[6]Thestudyofproteins,however,hasbeenascientificfocusforamuchlongertime.Studying
proteinsgeneratesinsightonhowproteinsaffectcellprocesses.Conversely,thisstudyalsoinvestigateshowproteinsthemselvesare
affectedbycellprocessesortheexternalenvironment.
Proteinsprovideintricatecontrolofcellularmachinery,andareinmanycasescomponentsofthatsamemachinery.[7]Theyservea
varietyoffunctionswithinthecell,andtherearethousandsofdistinctproteinsandpeptidesinalmosteveryorganism.Thisgreatvariety
comesfromaphenomenonknownasalternativesplicing,inwhichaparticulargeneinacell'sDNAcancreatemultipleproteintypes,
basedonthedemandsofthecellatagiventime.
Thegoalofproteomicsistoanalyzethevaryingproteomesofanorganismatdifferenttimes,inordertohighlightdifferencesbetween
them.Putmoresimply,proteomicsanalyzesthestructureandfunctionofbiologicalsystems.[8]Forexample,theproteincontentofa
cancerouscellisoftendifferentfromthatofahealthycell.Certainproteinsinthecancerouscellmaynotbepresentinthehealthycell,
makingtheseuniqueproteinsgoodtargetsforanticancerdrugs.Therealizationofthisgoalisdifficultbothpurificationand
identificationofproteinsinanyorganismcanbehinderedbyamultitudeofbiologicalandenvironmentalfactors.[9]
ProteomicsWorkflows
Thefirststepofproteomicsissamplepreparation.Inthisstep,wearetryingtoextractproteinfromcells.Inthesecondstep,weuse
methodssuchas2Delectrophoresistoseparatedifferentproteins.Thenwetrytocutproteinsintopeptidessincepeptidesareeasierto
detect.Intheforthstep,weusemassspectrometrytodetectpeptidesandpeptidesfragments.Finally,wecanthendeterminethesequence
oftheproteinbyinterpretingallthedataobtained.
BroadBasedProteomics
BecauseProteomicsisgrowingataveryrapidpace,thereisashiftinthefieldawayfromaspecialized/focusedwayofconducting
studiesandtowardsamoreglobalperspective.Broadbasedproteomicspresentsauniqueperspectiveonthefieldofproteomicsbecause
itallowsforonetotakeonthisgeneralperspectivebysettingouttounderstandtheproteomeasawhole.Acriticalaspecttothisstrategy
isplanningaheadandindoingso,themostappropriateplansandtechnologiescanbeimplementedinthemostefficientmanner.By
developingastrategytailoredtounderstandingaparticularproteome,problemsandsetbackscanbeavoidedduringthestudy.
Thefirststepwhenutilizingbroadbasedproteomicsistodevelopahypothesisspecifictotheproteomebeingstudied.Itisbesttochoose
organismsthatalreadyhaveagreatdealofgenomicinformationavailable,sincethegenomeisalwaysausefulsupplementtoproteomic
information.Oncetheahypothesisandorganismareestablished,thepropertechnologiesshouldbechosenandthesetechnologies
shouldbecompatiblewithwhateverbiologicalfactorsarepresent(i.e.sampletype).Someimportantandrelevantproteomicmethods
includeHPLC,MassSpectrometry,SDSPAGE,twodimensionalgelelectrophoresis,andperhapsinsilicoproteinmodeling.
Sincetherearemultitudesofsampletype,samplepreparation,andanalyticaltechnologycombinationspossible,itisobviouswhycareful
planningfromabroadbasedproteomicperspectiveiscritical.Byplanningupfront,anefficientproteomicstudycanbeconducted.And
whentheeffortsofmanybroadbasedproteomicstudiesaretakentogether,understandingtheproteomeinitsentiretybecomesarealistic
possibility.
References
1.^AmericanMedicalAssociation."Proteomics."http://www.amaassn.org/ama/pub/category/3668.html
2.^Hartl,DanielL.,Jones,ElizabethW."Genetics:AnalysisofGenesandGenomes".JonesandBartlettPublishers:Boston,2005.
3.^Weaver,RobertF."MolecularBiology,2ndEdition".McGrawHill:Boston,2002.
4.^Twyman,Richard."Proteomics."http://genome.wellcome.ac.uk/doc_wtd020767.html
5.^Colinge,JacquesandKeiryn
L.Bennett."Introductionto
ComputationalProteomics".
PLoSComputBiol.2007July
3(7):e114.
6.^"HistoryofProteomics."
AustralianProteomeAnalysis
Facility.
BroadbasedProteomicsApproachvstraditionalfocusedapproach
http://www.proteome.org.au/HistoryofProteomics/default.aspx
7.^Graves,P.R.,T.A.J.Haystead."MolecularBiologist'sGuidetoProteomics".MicrobiologyandMolecularBiologyReviews:
Vol.66No.1,2002.
8.^"ProteomicsOverview."http://www.proteomicworld.org/
9.^vanWijk,K.J."ChallengesandProspectsofPlantProteomics".PlantPhysiol.2001June126(2):501508.
ChapterWrittenbyJ.Reuter(Zel2008)andS.Lafergola(DieselSandwich)
ArticlesSummarized
AdvancesinProteomicWorkflowsforSystemsBiology
JohanMalmstrom,HookeunLee,andRuediAebersold.CurrOpinBiotechnol18(4):378384(2007)(http://www.pubmedcentral.nih.gov/
picrender.fcgi?artid=2048812&blobtype=pdf)
MainFocus
Thearticlesummarizesrecentimprovementsaswellassomeprincipallimitationsofshortguntandemmassspectrometrybased
proteomics.Furthermore,italsobrieflyintroducesstepsoftargeteddrivenquantitativeproteomics.
Summary
Inrecentyears,greatimprovementshavebeenmadeinallthepartsofnontargetedmassspectrometrybasedproteomicsincluding
samplepreparation,dataacquisition,dataprocessingandanalysis.Inthesamplepreparationprocess,withtheintroductionofIEF
separationmethod,resolutionobtainedfromclassicaltwodimensionalchromatographypeptideseparationisgreatlyimproved.
Improvementsarealsomadeinthefieldofdataqualitywhichisincreasedbythedevelopmentofhighlyreproduciblecapillary
chromatographymethodsandquantitativeanalysisbystableisotopelabelingmethod.Highmassresolutionandaccuracycouldbe
achievednowbydifferenttypesofmassspectrometrysuchasTOFTOF,QTOFinthedataacquisitionprocess.Furthermore,different
typesofmassanalyzersandionsourceshavebeencombinedtoincreasetheproteomecoverage.Withthedevelopmentofdatabasesearch
tools,thequalityofproteomicsdatacouldbemoreaccuratelyassessedandestimatedinthedataprocessingandanalysisprocess.
Despitealltheseimprovementsachieved,limitationsexistinshotgunapproaches.Forexample,shotgunMSdatasetsareextremely
redundantwhichgreatlyaffecttheidentificationofpeptidespresentinproteomicsamples.Theexistenceofsemitrypticornontryptic
peptidesinsamplesmadethesamplemorecomplex.Saturationeffectgreatlyreducesthediscoveryrateofnewproteins.Manypeptides
thatdetectedbyMassSpectrometrycouldnotbeidentified,makingitdifficulttocomparesampletosample.
Thelimitationsofshotgunapproachesmadethedevelopmentoftargeteddrivenquantitativeproteomicsnecessary.Thefirststepof
targeteddrivenquantitativeproteomicsisproteinandpeptideselection.Thisstepcouldbefinishedbothexperimentallyand
computationally.Forthemultiplereactionmonitoring(MRM)anddataanalysisstep,multiplereactionmonitoringwasappliedto
proteomicsdataanalysis.Relevancetothecourse:thissourceisabriefoverviewofrecentimprovementsintargetedmassspectrometry
(onemethodofproteomics)basedproteomicsaswellassomelimitations.Italsointroducedanotherfieldofproteomics:targeteddriven
quantitativeproteomics.
NewTerms
Electrosprayionization
Atechniqueusedinmassspectrometrytoproduceions.Itisespeciallyusefulinproducingionsfrommacromoleculesbecauseit
overcomesthepropensityofthesemoleculestofragmentwhenionized(http://en.wikipedia.org/wiki/Electrospray_ionization)
Matrixassistedlaserdesorption/ionization(MALDI)
Asoftionizationtechniqueusedinmassspectrometry,allowingtheanalysisofbiomolecules(biopolymerssuchas
proteins,peptidesandsugars)andlargeorganicmolecules(suchaspolymers,dendrimersandothermacromolecules),whichtendto
befragileandfragmentwhenionizedbymoreconventionalionizationmethods(http://en.wikipedia.org/wiki/MALDITOF)
PeptideAtlas
Amultiorganism,publiclyaccessiblecompendiumofpeptidesidentifiedinalargesetoftandemmassspectrometryproteomics
experiments(http://www.peptideatlas.org/)
Multiplereactionmonitoring
MRMexperiments,usingatriplequadrupoleinstrument,aredesignedforobtainingthemaximumsensitivityfordetectionoftarget
compounds.Thistypeofmassspectrometricexperimentiswidelyusedindetectingandquantifyingdruganddrugmetabolitesin
thepharmaceuticalindustry(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2291721)
FTICRmassspectrometry
Fouriertransformioncyclotronresonancemassspectrometry,alsoknownasFouriertransformmassspectrometry,isatypeof
massanalyzer(ormassspectrometer)fordeterminingthemasstochargeratio(m/z)ofionsbasedonthecyclotronfrequencyof
theionsinafixedmagneticfield(http://en.wikipedia.org/wiki/Fourier_transform_ion_cyclotron_resonance)
CourseRelevance
Thissourceisaboutnontargetedmassspectrometryandtargetedapproacheswhichareimportantmethodsintheidentificationof
proteins(animportantstepinproteomics).
BroadBasedProteomicStrategies:APracticalGuidetoProteomicsandFunctionalScreening
GrahamDavid,ElliotSteven,VanEykJennifer."JournalofPhysiology"563:19(2005)(http://jp.physoc.org/cgi/content/full/563/1/1)
MainFocus
Thisarticlesummarizeswhatbroadbasedproteomicsisandhowonecandesignastudyusingthisglobalviewstrategy.Itfirstbriefly
looksatthecurrenttechnologyinproteomicsandthendiscusseshowthesetechnologiescanbeincorporatedintoastudy.
Summary
Proteomicsasafieldisbecomingaverydauntingonetoenterbecausemanystudiesaregettinglostinthecomplicatedfocuseddetails.
Tohelpassistwiththischallenge,aresearchercanemploybroadbasedproteomics.Broadbasedproteomicsisastrategywherecareful
planningisemployedupfronttoansweraquestionaboutaproteome(forinstance,comparisonsbetweenatissueinadiseasedstateanda
normalstate)usingthemostappropriateandapplicabletechnologiesavailable.Bydevelopingastrategyatthebeginningofaproteomics
study,possiblesetbacksduringthestudyareavoided.
Thefirststepistodevelopageneralhypothesisthatisspecifictotheproblemorissuethatisbeingstudied.Sinceproteomicsmirrors
genomics,aproteomicstudyisincreasinglydifficultwhenthegenomeofthemodelorganismisn'tknown.Forthisreason,organisms
wherethemajorityofthegenomeisknown(80%orgreater)shouldbechosen.Onceaproperorganismhasbeenchosenforstudy,the
nextfactorstoconsiderarethetypeofdatathatwillbegeneratedandalsothesamplesource.Someproteomicmethodsyieldqualitative
data,whileothersyieldquantitativesothetypeofdataneededshouldbedeterminedbeforeamethodischosen.Atthesametime,the
sourceofthesampleisimportantindeterminingtheextractionandpurificationmethods.Typicalsampletypesinclude:urine,blood
(plasma/serum)andmucosalsecretions.Proteinconcentrationwithinthesampleisimportant,andoneshouldexpectreasonable
extractioniftheproteincanbevisualizedonacoomassiebluestainedgel(>300ng).Theseparationtechniquechosenshouldreflectthe
characteristicsoftheprotein(s)ofchoice(hydrophobicvshydrophilic,molecularmass,etc).
Anothermajorfactorintheplanningprocessisestimatingthedifficultyinthepreparationofthefractionedsampleformassspectrometry
identification.Eachmassspectrometrytechniquerequiresvaryingdegreesofpreparation,andsomearemuchmorecomplicatedthan
others(2DEwithMS/MSanalysisrequiresgreaterpreparationthanHPLCwithMS,forinstance).Sincemassspectrometryisoftenthe
stepwherealotofproteomicstudiesencounterdifficulty(bothinpreparationandininterpretationoftheresults),itisveryimportantto
chooseamethodthatisappropriatefortheproteinsample.
Withtheadventofproteomicdatabasesinrecentyears,bioinformaticshashadanincreasingpresenceinproteomicstudies.Forthis
reason,almostallproteomicstudiesshouldincorporatebioinformaticsandconsequentlyit'simportantfortheresearchteamtohave
somebioinformaticsknowledge.Anddependingonhowmuchdatawillbereceivedattheendofthestudy(dependingontheanalysis
methodschosen),theresearchteamcandeterminehowmuchbioinformaticanalysisshouldbeneeded.
Afinalfactortoconsideriswhethertobringinoutsideassistanceortoattemptthestudyinamoreselfcontainedway.Keepingitself
containedallowsfortheresearchteamtokeepitsdataintegratedandalsokeepsmiscommunicationtoaminimum.Bringinginoutside
help,ontheotherhand,couldallowaresearchertotackleproblemsthatwouldbelargeandnormallynotsolvablewithasmallerteam.
Whilebringinginoutsideassistanceseemspromising,it'simportanttonotlosecontroloverthedataandtomakesurethattheteamisnot
spreadouttryingtoaccomplishmorethanitcanhandle.
Sincetherearemanywaystostudyacell'sproteome,carefulplanningshouldbeimplementedatallstagesofaproteomicsstudy.
Throughbroadbasedproteomics,aresearchercandefineatestplanbeforeanyactualstudyisperformed.Andwhenusedappropriately,
thisstrategycanleadtoproductiveandefficientprojectsthatwillbringscienceonestepclosertounderstandingtheproteomeasawhole.
NewTerms
Isoforms
Asetofdifferentproteinsthatformbecauseofsinglenucleotidepolymorphismsinthegenomicsequence.(
http://en.wikipedia.org/wiki/Isoform)
Singlenucleotidepolymorphism(SNP)
aDNAsequencevariationoccurringwhenasinglenucleotideinthegenomediffersbetweenmembersofaspecies.(
http://en.wikipedia.org/wiki/Single_nucleotide_polymorphism)
Posttranslationalmodification(PTM)
thechemicalmodificationofaproteinafterithasbeentranslated.Itisusualyoneofthelaststepsinproteinbiosynthesisformost
proteins.(http://en.wikipedia.org/wiki/Posttranslational_modification)
Subproteome
asubfractionedsubsetoftheproteome.Oftenthesearelinkedtoareaofthecell(organelleforinstance)orbychemicalproperties.
Peptidemassfingerprinting(PMF)
ananalyticaltechniqueforproteinidentification.Theunknownproteinofinterestisfirstcleavedintosmallerpeptidesandafter
massisdeterminedusingmassspectrometry,theirmassesarecomparedtoeitheradatabasecontainingknownproteinsequencesor
agenome.(http://en.wikipedia.org/wiki/Peptide_mass_fingerprinting)
CourseRelevance
Thisarticleisrelevantbecauseaglobalviewofproteomicsisbecomingmoreimportant.Asthewealthofinformationabout
proteinsexpands,understandingtheproteomefromabroadviewpointisbecomingmoreandmoreuseful.
WebsitesSummarized
TheAssociationofBimolecularResourceFacilities:ProteomicsResearchGroup(PRG)
Websitecommittee:PamelaScottAdams,MichelleDetwiler,DavidMohrJamesEe,Dr.XiaologYang,Dr.LenPackman,Dr.Anthony
Yeung,http://www.abrf.org/index.cfm/group.show/Proteomics.34.htm#R_3(3/25/09)
MainFocus
ThiswebpageisabouthowtheAssociationofBimolecularResourceFacilitiesrelatestoproteomics.Ofparticularimportanceisthe
ProteomicsResearchGroupwithintheABRF.
Summary
TheAssociationofBimolecularResourceFacilities(ABRF)isaninternationalassociationofresearchfacilitiesandlaboratoriesthatis
focusedoncoreresearchinBiotechnology.Theassociationencouragesthesharingofinformationthroughconferences,aquarterly
journal,andgroupstudies.TheABRFhasaheavyinfluenceonthefieldofproteomics,andtherearefivemainresearchgroups(RG)that
dealwithproteomicsinsomeway:ProteinExpression(PERG),ProteinSequencing(PSRG),ProteinInformatics(iPRG),Proteomics
(PRG),andProteomicsStandards(sPRG).
Ofparticularimportance,theProteomicsResearchGroupallowsforresearchersthroughouttheworldinthefieldofproteomicstoshare
theirproteinanalysisinformationfreely.Obviously,sinceunderstandingtheproteomeisaboutbringingtogetherinformationonmany
differentproteins(whichisinformationthatrequiresagreatamountofeffort/time/moneytoachieve),thesharingof
protein/subproteomicinformationisimperativetobeginningtounderstandaproteomeinitsentirety.Thiswebsitehasnumerouslinksto
studiesperformedbyresearchgroupsthroughouttheworld.
NewTerms
DeNovoPeptideSequencing
Peptidesequencingthatisperformedwithoutanypriorknowledgeoftheaminoacidsequence.
(http://www.ionsource.com/tutorial/DeNovo/DeNovoTOC.htm)
QuantitativeProteomics
Hasthegoalofobtainingquantitativeinformationaboutalltheproteinsinaparticularsample.Thisisusefulbecauseitallowsfor
onetoseethedifferencesinproteinsamples.(http://en.wikipedia.org/wiki/Quantitative_proteomics)
CourseRelevance
ThisisanoverviewoftheAssociationofBiomolecularResourceFacilities(ABRG)andhowitrelatestoproteomics.Thereisa
greatdealofrelevantinformationonthiswebsitethatthoseinproteomicswillfinduseful.
Writer/Producer:RickGroleau,SubjectMatterExpert:HannoSteen,PhD,Designer:PeggyRecinos,Developer:JeffreyTesta,
http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.html(28March2009)
MainFocus
Thiswebpageisabouttheimportanceandchallengesinproteomics.Italsointroducesmajorstepsof
proteomicsbriefly.
Summary
Proteomicsisimportantforustounderstandbiologicalprocessessinceallthefunctionsareaccomplished
byproteinsincell.Butasthenumberofproteinsaresolargeandaminoacids(whichareunitsofprotein)
aresosmall,thestudyisquitechallenging.Therearefivestepstoanalyzeproteinsequences:sample
preparation,separation,ionization,massspectrometryandinformatics.Firstofall,weobtaincellsandextract
proteinsfromthecells.Thenweusemethodssuchas2Delectrophoresistoseparateproteins.Next,weuse
proteasetocutproteinsintopeptides.Massspectrometryallowsustoidentifyindividualpeptidesaswellas
peptidesfragments.Finally,byinterpretingthedata,weareabletodeterminethesequenceofproteins.
NewTerms
Biopsy
Abiopsyisamedicaltestinvolvingtheremovalofcellsortissuesforexamination.Itistheremoval
oftissuefromalivingsubjecttodeterminethepresenceorextentofa
disease(http://en.wikipedia.org/wiki/Biopsy)
TOF
Thetimeofflight(TOF)describesthemethodusedtomeasurethetimethatittakesforaparticle,objectorstreamtoreacha
detectorwhiletravelingoveraknowndistance(http://en.wikipedia.org/wiki/Timeofflight)
Quadrupolemassspectrometry
Thequadrupolemassanalyzerisonetypeofmassanalyzerusedinmassspectrometry.Itconsistsof4circularrods,setperfectly
paralleltoeachother.Inaquadrupolemassspectrometerthequadrupolemassanalyzeristhecomponentoftheinstrument
responsibleforfilteringsampleions,basedontheirmasstochargeratio(m/z).Ionsareseparatedinaquadrupolebasedonthe
stabilityoftheirtrajectoriesintheoscillatingelectricfieldsthatareappliedtothe
rods(http://en.wikipedia.org/wiki/Quadrupole_mass_analyzer)
Electronsprayionization
Electrosprayionization(ESI)isatechniqueusedinmassspectrometrytoproduceions.Itisespeciallyusefulinproducingions
frommacromoleculesbecauseitovercomesthepropensityofthesemoleculestofragmentwhen
ionized(http://en.wikipedia.org/wiki/Electrospray_ionization)
Dalton
Daltonistheunitofmeasurementforatomicmass.OneDaltonisequalto1/12ththemassofoneatomof
carbon12(http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P1.html)
CourseRelevance
Thisisanoverviewofproteomics.Itsummarizestheproceduresandimportanceofproteomicsverybriefly.
InstituteofBiologyandMedicalGeneticsoftheFirstFacultyofMedicineofCharlesUniversityandtheGeneralTeaching
Hospital,http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm(6April2009)
MainFocus
Thiswebsitediscussestheaimsanddefinitionsofproteomics.Italsointroducestwoimportant
methodsinproteomcisstudies2Dproteinelectrophoresisandmassspectrometryaswellas
proteomicsinmedicine
Summary
Proteomicsisabroadfieldwhichincludesexpressionproteomics,proteindistributionin
subcellularcompartmentsoftheorganelles,posttranslationalmodificationsofthe
proteins,structuralproteomicsandfunctionalproteomics,clinicalproteomicsandsoon.Even
thoughanalysisoftheexpressionontranscriptslevelispossiblewiththeintroductionofRNA/cDNAmicroarray,proteomicsisstill
importantsincenotallmRNAwillbetranslatedandtheprocessessuchasRNAsplicing,posttranslationalproteinmodificationsexist.
Twodimensional(2D)proteinelectrophoresisiscommonlyusedtoseparateproteinsbasedontheirPIandmass.Massspectrometryisan
importantmethodinproteomicssinceitcannotonlybeusedforproteinidentificationbutcanalsobeusedforproteinposttranslational
modificationanalysis.
Oneofthemajorapplicationofproteomicsinmedicineistheidentificationofmarkersinallthestepstotreatdiseases.Otherapplications
includedrugdiscoveryandpharmacoproteomics.
NewTerms
HumanProteomeOrganization(HUPO)
TheHumanProteomeOrganisation(HUPO)isaninternationalscientificorganizationrepresentingandpromotingproteomics
throughinternationalcooperationandcollaborationsbyfosteringthedevelopmentofnewtechnologies,techniquesand
training(http://www.hupo.org/)
structuralproteomics
Structuralproteomicsisaninternationalcollaborationprojectforsolving3Dproteinstructuresataproteome
scale(http://en.wikipedia.org/wiki/Structural_proteomics)
SwedishHumanProteinAtlas
TheSwedishHumanProteinAtlasprogram(HPA),fundedbythe(nonprofit)KnutandAliceWallenbergFoundation,invites
submissionofantibodiesfrombothacademicandcommercialsourcestobeincludedinthehumanproteinatlas
(http://www.proteinatlas.org)
Posttranslationalmodification(PTM)
Posttranslationalmodification(PTM)isthechemicalmodificationofaproteinafteritstranslation.Itisoneofthelaterstepsin
proteinbiosynthesisformanyproteins(http://en.wikipedia.org/wiki/Posttranslational_modification)
Isoelectricpoint
IsoelectricpointissuchapHvalue,wheretheoverallproteinchargeequalsto
zero(http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm)
CourseRelevance
Thiswebsitegivesbriefdefinitionandaimsofproteomics.Italsointroducesprinciplesof2Delectrophoresisandmass
spectrometrywhichareimportantmethodsinproteomics.
Contact:jxr0084@rit.edu,sfl9376@rit.edu
References
JohanMalmstrom,HookeunLee,andRuediAebersold."AdvancesinProteomicWorkflowsforSystemsBiology"(http://www.pub
medcentral.nih.gov/picrender.fcgi?artid=2048812&blobtype=pdf)CurrOpinBiotechnol18(4):378384(2007)
RickGroleau,HannoSteen,PeggyRecinos,JeffreyTesta."IntroductiontoProteomics"
http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.html(28March2009)
InstituteofBiologyandMedicalGeneticsoftheFirstFacultyofMedicineofCharlesUniversityandtheGeneralTeaching
Hospital."IntroductiontoProteomics"http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm(06April2009)
ProteinSamplePreparation
Presentation
Printversion
Introduction
Astechnologicaladvancesaremadeinthefieldofproteomics,itisseenthatadvancesarenecessaryinthepreparationofproteinsamples
priortoanyparticularprocedure.Anumberofissuesariseinthisrespectincludingsamplecleanup,fractionation,enrichment,andthe
alsosampleconditionoptimization.Considerationsofthisnaturecanbecrucialinobtainingrelevantresultsfromanexperimentsomeso
muchsothatexpertsfeelthefieldofproteomicsiscurrentlybeinglimitedbythelackofsignificantadvancementinsamplepreparation
techniques.
Thisfacetofproteomicsisbecomingparticularlycriticalinthecaseofhighthroughputprotocolswherethenecessaryconditionsofa
sampleinonestagemaydirectlyconflictwiththeefficacyofasecondstage.Forexample,duringtheinitialstepin2Delectrophoresis,
isoelectricfocusing,allproteinsinasamplearegivenanetchargeofzerowhilethesecondstep,gelelectrophoresis,requiresanegative
chargeonallproductsinthesampleinordertoinducemovementthroughthegelmatrix.
Manycompaniesofferprepackagedkitsthatwillallowyoutopreparesamplesformanydifferenttechniques.Theyalsooffermany
proteinsamples,andotherproteintechnologies.Manyofthesecompaniesarealsoontheforefrontofproteinanalysistechnology.Some
examplesare:
1.www.millipore.com(http://www.millipore.com/immunodetection/id3/immunodetectionresearchhome&cid=BIOSSWIKI100311
07SP)
2.www.biorad.com(http://www.biorad.com)
3.www.qiagen.com(http://www.qiagen.com)
4.www.gelifesciences.com(http://www.gelifesciences.com/aptrix/upp01077.nsf/content/trap_site)
5.www.invitrogen.com(http://www.invitrogen.com)
6.www.eksigent.com(http://www.eksigent.com)
7.www.agilent.com(http://www.agilent.com)
8.www.beckmancoulter.com(http://www.beckmancoulter.com)
9.www.caliperls.com(http://www.caliperls.com)
10.www.glygen.com(http://www.glygen.com)
11.www.piercenet.com(http://www.piercenet.com)
12.www.bioproximity.com(http://www.bioproximity.com/content/guideproteomicsamplepreparation)
ThissectionispartofanongoingprojectattheRochesterInstituteofTechnology(http://www.rit.edu),involvingtheBioinformatics(htt
p://bioinformatics.rit.edu/)department.CurrentlytheprojectisbeingworkedonbyaProteomicsClasstaughtbyDr.PaulCraig.
Bibliography
JrgvonHagen,VCHWiley2008ProteomicsSamplePreparation.ISBN9783527317967
PlantProteomicsaboutTwoDimensionalGelElectrophoresis
Presentation
Printversion
Thispagecontainsprotocolsthatarefrequentlyusedinproteomics.Youarewelcometoaddprotocolsthischapter.
1.PlantProteomicsaboutTwoDimensionalGelElectrophoresis
ProteinSeparationsChromatography
Presentation
Printversion
Chapterwrittenby:LauraGrellandAlexanderButarbutar
Contactllg3875@rit.eduornbb3924@rit.eduforcontributions
ChaptermodifiedbyKaiBurnettandDaliaGhoneim
Contactkab9783@rit.eduordxg6098@rit.edu
Introduction
(Res1)(http://www.rit.edu/~pac8612/webionex/website/html/ione7dim.html)
Toobtainapureproteinsample,aproteinmustbeisolatedfromallother
proteinsandcellularcomponents.Thiscanprovetobeadifficulttaskasa
singleproteinoftenmakesuponly1%ofthetotalproteinconcentrationofa
cell.Therefore99%oftheproteincomponentsofasamplemustberemoved
beforeitcanbeclassifiedaspure.Ataskthatisequallychallengingis
keepingtheproteininitsactiveform.Whenwepurifyproteinsweremove
themfromtheirnaturalenvironments.Asaresult,itisnecessarytosimulate
thepH,saltconcentrationandreducingconditionsinwhichtheyarenormally
found.Intheprocessofobtaininganactiveandpuresamplewewantto
minimizethenumberofstepstakeninordertomaximizetheyieldattheend
oftheseparation.Finally,sinceproteinsaremadewiththeintentionofonly
functioningforashortperiodoftime,itisalsocriticaltoobtainoursampleas
quicklyaspossible.Allthesecomponentsofproteinseparationscanbe
successfullyachievedbyagroupofseparationmethodscollectivelyknownas
chromatography.(http://fig.cox.miami.edu/~cmallery/255/255tech/ecbxpanel HPLC
4x4.pdf)
Thereareseveralpropertiesofproteinsthatcanbetakenadvantageoftoseparateproteins.Differenttypesofchromatographytake
advantageofdifferentproperties.Proteinscanbeseparatedby:
size
shape
hydrophobicity
affinitytomolecules
charge
Inthischapterseveraldifferentchromatographicmethodswillbe
introducedanddescribed.Whilethemethodsoutlinedbelowallusedifferent
characteristicsofproteinstoseparateproteinsfromoneanother,theyall
utilizeaninsolublestationaryphaseandamobilephasethatpassesoverit.
Themobilephaseiscommonlyaliquidsolution.Itcontainstheproteinwe
wanttoisolate.Thestationaryphaseontheotherhandismadeupofa
groupingofbeads,usuallybasedonacarbohydrateoracrylamidederivative,
thatareboundtoionicallychargedspecies,hydrophobiccharacters,or
affinityligands.Muchofthesuccessofchromatographyisassociatedwith
theselectionofanappropriatestationaryphase.
Incolumnchromatography(http://fig.cox.miami.edu/~cmallery/255/255his
t/ecbxp4x3_chrom.jpg),whenaproteinsampleisappliedtothecolumn,it ATypicalColumn
equilibratesbetweenthestationaryphaseandthemobilephase.Dependingon
thetypeofchromatography,proteinswithcertaincharacteristicswillbindto
thestationaryphasewhilethoselackingthesoughtcharacteristicswillremaininthemobilephaseandpassthroughthecolumn.For
exampleinionexchangechromatography,apositivelychargedproteinwouldbindtoanegativelychargedstationaryphase,whilethe
negativelychargeproteinwillbeelutedfromthecolumnwiththemobilephase.Thefinalstepinvolvesdisplacingtheproteinfromthe
stationaryphase,alsoknownaselution,byintroducingaparticlewhichwillcompetewiththeproteinbindingsiteonthestationary
phase.Todayvariouscommercialcolumnarereadilyavailable,specificallyBioRad(http://www.biorad.com/B2B/BioRad/product/br_ca
tegory.jsp?BV_SessionID=@@@@0655054085.1146799058@@@@&BV_EngineID=cccfaddhjklfdfkcfngcfkmdhkkdfll.0&categoryPa
th=%2fCatalogs%2fLife+Science+Research%2fChromatography+%7c+Protein+Purification%2fChromatography+Columns&divName=
Life+Science+Research&catLevel=4&lang=English&country=null&loggedIn=null&catOID=31031&isPA=false&serviceLevel=null),
SigmaAldrich(http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Proteomics_and_Protein_Expr_/Protein_Analysis/Chromat
ography.html),GEHealthcare(http://www4.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_Prod~SelGuides~Media)
andOmnifit(http://www.chromtech.net.au/glass_columns.cfm)offersawidevarietyofchromatographycolumn.
Theimageaboveisachromatogramthatshowstheresultsofaseparationbasedonsignalsinterpretedbyadetector.
tmthetimerequiredforthemobilephasetotraveltheentirelengthofthecolumn
trthetimerequiredforaspecificproteintoelutefromthecolumn
Resources
1.Craig,P.DesigningaSeparation(http://www.rit.edu/~pac8612/webionex/website/html/ione7dim.html)
2.FloridaStateUniversity,"Chromatography"MichaelBlaber'sBiochemistryLab(http://wine1.sb.fsu.edu/bch4053l/Lecture04/Lectu
re04.htm)
3.GEHealthcare[10](http://www.gelifesciences.com)[11](http://www.gelifesciences.com/proteinpurification?)
4.BioPharmInternationalGuideBasicsofChromatography(http://www.biopharminternational.com/biopharm/data/articlestandard/bi
opharm/522003/80026/article.pdf)(2003).
5.BioRadChromatographyProteinPurification(http://www.biorad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@
@@0575591939.1146688325@@@@&BV_EngineID=cccfaddhjjigfilcfngcfkmdhkkdfll.0&divName=Life+Science+Research&c
ategoryPath=%2fCatalogs%2fLife+Science+Research%2fChromatography+%7c+Protein+Purification&loggedIn=false&serviceLe
vel=Lit+Request&lang=English&country=null&catLevel=3&catOID=30719&isPA=false)||GreenFluorescentProtein
ChromatographyKit(http://www.biorad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@0641748830.114679
8866@@@@&BV_EngineID=cccfaddhjklfdfkcfngcfkmdhkkdfll.0&divName=Life+Science+Education&categoryPath=%2fCatal
ogs%2fLife+Science+Education%2fClassroom+Kits%2fSize+Exclusion+Chromatography+Kit&loggedIn=false&lang=English&c
ountry=null&catLevel=4&catOID=18885&isPA=false&serviceLevel=Lit+Request)
6.BioForumTopicsInChromatography(http://www.forumsci.co.il/HPLC/topics.html)
7.JournalofChromatographicScience(http://www.jchromsci.com/)
8.M.IsabelPedrazaMayerChromatographyDatabase(http://www.chromatographydb.com/)
References
1.Harris,D.C."QuantitativeChemicalAnalysis6thEdition",W.H.FreemanandCompany:NewYork..
2.PatrickMcKayAnIntroductiontoChromatography(http://www.accessexcellence.org/LC/SS/chromatography_background.html)
SeniorResearchAssociate,DepartmentofRecoverySciences,Genentech,Inc*.
.*DenotesFreeArticle
ProteinSeparationsElectrophoresis/Introductionto
Electrophoresis
Presentation
Printversion
IntroductiontoElectrophoresis
Definitions
electrophoresis(lk'trfr'ss)n.[12]
1)Themigrationofchargedcolloidalparticlesormoleculesthroughasolutionundertheinfluenceofanappliedelectricfieldusually
providedbyimmersedelectrodes.Alsocalledcataphoresis.
2)Amethodofseparatingsubstances,especiallyproteins,andanalyzingmolecularstructurebasedontherateofmovementofeach
componentinacolloidalsuspensionwhileundertheinfluenceofanelectricfield.
analyte(anlt)n.[13]
Achemicalsubstancethatisthesubjectofchemicalanalysis.
ElectrophoresisTheory
Separationbyelectrophoresisdependsondifferencesinthemigrationvelocityofionsorsolutesthroughagivenmediuminanelectric
field.Theelectrophoreticmigrationvelocityofananalyteis:
whereEistheelectricfieldstrengthand istheelectrophoreticmobility.
Theelectrophoreticmobilityisinverselyproportionaltofrictionalforcesinthebuffer,anddirectlyproportionaltotheionicchargeofthe
analyte.Theforcesoffrictionagainstananalytearedependentontheanalyte'ssizeandtheviscosity()ofthemedium.Analyteswith
differentfrictionalforcesordifferentchargeswillseparatefromoneanotherwhentheymovethroughabuffer.AtagivenpH,the
electrophoreticmobilityofananalyteis:
whereristheradiusoftheanalyteandzisthenetchargeoftheanalyte.
Differencesinthechargetosizeratioofanalytescausesdifferencesinelectrophoreticmobility.Small,highlychargedanalyteshave
greatermobility,whereaslarge,lesschargedanalyteshavelowermobility.Electrophoreticmobilityisanindicationofananalyte's
migrationvelocityinagivenmedium.Thenetforceactingonananalyteisthebalanceoftwoforces:theelectricalforceactinginfavor
ofmotion,andthefrictionalforceactingagainstmotion.Thesetwoforcesremainsteadyduringelectrophoresis.Therefore,
electrophoreticmobilityisaconstantforagivenanalyteunderagivensetofconditions.[14]
ApplicationsofElectrophoresis
Electrophoresishasawidevarietyofapplicationsinproteomics,forensics,molecularbiology,genetics,biochemistry,andmicrobiology.
Oneofthemostcommonusesofelectrophoresisistoanalyzedifferentialexpressionofgenes.Healthyanddiseasedcellscanbe
identifiedbydifferencesintheelectrophoreticpatternsoftheirproteins.Proteinsthemselvescanalsobecharacterizedinthisway,and
somesenseoftheirstructurecanbederivedfromthemassesoffragmentsinsidethegel.[15]
Therearemanydifferenttypesofelectrophoresis,andeachcanbeusedforsomethingdifferent.Twodimensional(2D)electrophoresis,
forexample,hastheabilitytodiscernmanymoreproteinsthanmostofitscontemporaries.Manyofthesemethodswillbediscussedin
detailthroughoutthischapter.
References
1.^TheAmericanHeritageDictionaryoftheEnglishLanguage,FourthEdition.http://www.bartleby.com/61/
2.^TheMerriamWebsterOnlineDictionary.http://www.mw.com
3.^Mans,Andreasetal.BioanalyticalChemistry.ImperialCollegePress,2004.
4.^Twyman,Richard."Twodimensionalpolyacrylamidegelelectrophoresis."http://genome.wellcome.ac.uk/doc_wtd021045.html
Nextpage>
MicrofluidicElectrophoresis
WhatWillProteomicsGainfromMicrofluidic?
Proteomicscontributessignificantlytothediscoveryofproteinsandtheirfunctionsthatinfluencethebehaviorsofanorganism.Recent
studieshavefocusedonitsrolestoexploretheproteinsinasinglecellandtissuelevelastheyrepresentafingerprintforeachindividual
especiallyintermsofhowadiseaseexhibits.[1]Duetoalimitedamountofsamplefromasinglecellortissue,thestudyofproteomein
theselevelsbecomesabigchallengeforaregularbenchtopinstrument.Tofacilitatethislevelofstudy,amicrofluidictechnologyis
introducedanddevelopedintoanessentialtoolforproteomics.
Inadditiontothecapabilitytohandlesmallsample,themicrofluidictechnologyplaysanimportantroleinminiaturizingtheentire
system.Asaresult,abetterperformanceintermsoflessmaterialconsumption,fasterprocessingtime,moreautomated,andlowercost
canbeachieved.Anotheradvantageinsuchmicroscaleisthatthemixingbetweenthesampleandreagentsbecomesmoreeffective.[2]A
certainchemicalprocessthatusuallytakeshourscanbecompletedinminutes.Thisbenefitallowsthemicrofluidicbasedimmunoassay
tobeusedtomonitortheprogressofdisease.[1]And,withareductionincost,themicrofluidicdevicebecomesaperfectcandidatefor
manypointofcareapplications.[3]
Oneofthemostintriguingfeaturesprovidedbythemicrofluidictechnologyisitshighlyintegratedcapabilitywithothersystems
especiallywithmassspectrometry.[4]Multiplexingofvariousassaysisanotherexample.Thismultiplexingpotentialmakesmicrofluidic
baseddevicesahighthroughputsolutionin(bio)chemistryandbiomedicine.[5]
ElectrophoresisinMicrofluidicMicrosystems
Incorporatingbiotechnologywithmicrofluidicmakesamanipulationofverysmallvolumeofbiologicalfluidnotonlyfeasible,butalso
effectiveespeciallybymeansofelectrophoresis.Recentresearchanddevelopmenteffortshavebeenfocusedoninventingan
electrophoreticmicrosystemthatisfullyautomated,easytocustomizeforaspecificneed,andprovidestheresultsconsistentwiththe
goldstandard.Thismicrofluidicmicrosystemisusuallyreferredtoasalabonachip.Amongthemicrofluidicmicrosystemsusedinthe
analytical(bio)chemistry,themostwidelyusedmethodstocontrolthetransportofbiomoleculesoranalytesareeithergelorcapillary
electrophoresis.
Eventhoughbothtechniquesutilizethefactthatbiomoleculessuchasproteins,peptides,andDNAbecomechargedinabuffer,the
microfluidicgelelectrophoresisoperatesdifferentlyfromitscapillarycounterpartintermsoffluiddynamic.Inmicrofluidicgel
electrophoresis,thepresenceofporousgelmediumpreventsabulkflowinamicrofluidicchannel.[3]Ontheotherhand,thebulkflow
becomesanengineeringfactorthatinfluencestheelectrokineticsoftheanalytesasthepresenceofelectroosmoticflowneedstobe
consideredaswellinmicrofluidiccapillaryelectrophoresis.[6][7]Intheanalyticalviewpoint,theirelectrophoreticseparationmethodsare
alsodifferent.Thegelbasedelectrophoreticseparationofbiomoleculesisbasedonthedifferenceinsizeormolecularweight,whilethe
capillarybasedelectrophoresisseparationoperatesbytakingtheadvantageofthedifferenceinchargetomassratioamongbiomolecules.
Incomparisontothegoldstandardmethods,theanalyticalresultsobtainedfromthemicrofluidicgelandcapillaryelectrophoresisare
consistentwiththosefromthetraditionalslabgel[8]andcapillaryelectrophoresis[5],respectively.Inthedesignandapplication
viewpoints,however,therearesomeadvantagesanddisadvantagesofthesetwomethodsneededtobeconsidered.Intermsof
engineeringdesign,themicrofluidicgelelectrophoreticsystemismucheasiertocustomizeforspecificapplicationsandmultiplexing
sincenoinfluencefromthebulkflowneedstobeconsidered.Thismakesitmorestraightforwardtointegrateextrafeatureslikesample
preprocessingintothemicrofluidicgelelectrophoresis.[3]Nevertheless,sincethegelsievingmediumisnotrequiredinthecapillary
basedmicrosystem,thereusabilityofthemicrofluidiccapillaryelectrophoreticdeviceismuchhigherthanthegelbasedcounterpart.In
termsofbio(analytical)applications,themicrofluidiccapillaryelectrophoresishasadrawbacksuchthatitoperatespoorlytoanalyzethe
chargedparticlesofsimilarchargetomassratios.[3]Itisworthnotingthatsomemicrofluidiccapillaryelectrophoreticsystemiscapable
ofdirectinterfacewithmassspectrometry.[5]
Inthischapter,thedevicefabricationprocess,basicprincipleofoperation,andsomeclinicalapplicationsofbothmicrofluidicgeland
capillaryelectrophoresisaredescribedindetail.Eventhoughthischapterisdedicatedmainlytothemicrofluidicelectrophoresis,the
integrationofadditionalfeatureslikesamplepreprocessing,detection,andquantificationprocessesarealsoincluded.Unquestionably,
thisintegrationismadepossiblebythemicrofluidictechnology.
MicrofluidicGelElectrophoresis
Utilizingmicrofluidictechnologyingelelectrophoresisprovidesseveraladvantagestothestudyofproteomeinmanywaysthatcannot
beachievedbytheconventionalmethods.Fasterprocessingtime,moresensitivedetection,moreautomatedoperation,andhighly
integratedsystemarethemajorbenefitsofusingmicrofluidic.Inaddition,themicrofluidictechnologyallowsgelelectrophoreticsystem
tobeeasilycustomizedforaspecificapplication.Forexample,amicrofluidicgelelectrophoreticsystemcanbedesignedsuchthatoff
chipprocessingcanbeeliminated.Infact,itcanbeintegratedintothemicrofluidicbasedsystem.Theintegrationofsamplepreparation
isoneofthepracticalexamplesthatnotonlysimplifytheexperimentprotocol,butalsoimprovethedetectionsensitivity.[1]
Thissectionisdedicatedtoacustomizedmicrofluidicgelelectrophoresisforimmunoassayapplications.[3]Theimportantaspectssuchas
thefabricationprocess,principleofoperation,andclinicalapplicationswillbediscussedindetail.
I.FabricationProcess
Gelelectrophoresiscanbecustomizedforaspecificanalyticalstudysuchasanimmunoassaybyusingmicrofluidictechnology.The
customizedfabricationprocessofmicrofluidicgelelectrophoreticimmunoassaytobedescribedbelowisbasedonthedeviceusedby
Herretal.[1][3]Intheirstudy,anantibodywasusedasareportertodetectthepresenceofaparticularproteinasanantigen,potentiallya
diseasebiomarker.Astepbystepfabricationprocessisdescribedasfollows.
Step1:FabricationofSizeExclusionMembrane
Materials:
1.Degassed22%(15.7:1)acrylamide/bisacrylamide(6%bisacrylamidecrosslinker)
2.0.2%(w/v)VA086[9](photoinitiator)
3.1XTris/glycinebuffer
Thefirstcomponentofthemicrofluidicgelelectrophoreticimmunoassaytobefabricatedisasize
exclusionmembrane.Thismembraneisusedtoenhanceanalyteconcentration,thusimproving
sensitivityofdetection.Apolyacrylamidegelisutilizedtofabricatethismembrane.Itisdesigned
suchthatthepolyacrylamidegelhasaporesizesmallenoughforselectivelyallowinganalyte
moleculessmallerthan10kDatopassthrough.Thefabricationprocessbeginswithpatterninga Step1:Fabricationofsize
glasssubstratetocreatemicrofluidicchannelsandchambers.Thisprocessiscarriedoutbyusing
exclusionmembrane [3]
regularphotolithographyandwetetch.Holesaredrilledonthetopglasscover.Bothglasssubstrate
andcoverarethenbondedtogetherbyanodicbonding.Afterthemicrofluidicstructureiscreated,a
solutionofdegassed22%(15.7:1)acrylamide/bisacrylamide(6%bisacrylamidecrosslinker)and0.2%(w/v)VA086isintroducedinto
thechannel,asshowninthediagram.Asyringecanbeusedtoloadthesolutionintothechannel.Thegelprecursorsolutionisleftfor
equilibrationforapproximately30minutes.
Thesizeexclusionmembraneisfabricatedusinglaserphotopolymerization.A355nmUVlasersheetisusedtopatternthe
polyacrylamidegelatthespecifiedlocation,showninthediagram,tocreatethemembraneprofile.Thepolyacrylamidegelmembraneis
exposedtothelaseruntilpolymerized,whichtakesapproximately15seconds.Theremaininggelsolutionisvacuumedout,andthe
channelsarethencleanedbyrinsingwithbuffer.
Step2:FabricationofSeparationChannel
Materials:
1.Degassed8%(37.5:1)acrylamide/bisacrylamide(2.6%bisacrylamidecrosslinker)
Afterthesizeexclusionmembraneisfabricated,thenextstepistoconstructaseparationchannel.
Thisseparationchannelistheplacewhereproteinseparationtakesplace.Itcontainsamedium
porositypolyacrylamidegel.Likethemembrane,theseparationgelisfabricatedbyphoto
polymerization.Tofabricatetheseparationchannel,theseparationgelprecursorsolutioniscarefully
loadedintothemicrofluidicchannelbyasyringe.Thegelsolutioniscomposedofthedegassed8%
(37.5:1)acrylamide/bisacrylamide(2.6%bisacrylamidecrosslinker),0.2%(w/v)VA086 Step2:Fabricationofseparation
photoinitiator,and1XTris/glycinebuffer.Thegelloadingdirectionisindicatedbythearrowshown channel [3]
inthediagram.Thegelisloadeduptothespecifiedlocationtodefinetheseparationchannel.
Geluniformityisaveryimportantfactorfortherepeatabilityintheanalysis.Toguaranteeuniformity,allmicrofluidicchannelsonthe
separationsideofthemembranemustbefilledwithgelbeforepolymerization.Therefore,agelplugiscreatedtopreventthegelleakage
duringthesubsequentgelloading.Asshowninthediagram,thegelplugisfabricatedbyphotopolymerizationsuchthattheareanotto
bepolymerizedisprotectedbyadarkfieldmask.Usually,thephotopolymerizationprocesstakesabout10minutesusinga100WattUV
source.
Step3:FabricationofLoadingChannel
Materials:
1.Degassed3.5%(37.5:1)acrylamide/bisacrylamide(2.6%bisacrylamidecrosslinker)
File:UCEIStep3.png
Step3:Fabricationofloading
channel [3]
Inthisstep,theremainingmicrofluidicchannels(withoutgel)ontheseparationsideofthemembrane
arecarefullyfilledwithgelprecursorsolutionasindicatedbythearrowshowninthediagram.The
solutioncontainsthedegassed3.5%(37.5:1)acrylamide/bisacrylamide(2.6%bisacrylamidecrosslinker),0.2%(w/v)VA086
photoinitiator,and1XTris/glycinebuffer.Thisgelsolutionwillgenerateapolyacrylamidegelwithlargeporesizeanddefinetheloading
channels.
Afterbothseparationandloadingchannelsarefilledwithgel,thewholemicrofluidicdeviceisexposedtoUVfor15minutes.Asa
result,theseparationandloadinggelsarepolymerizedanddefineseparationandloadingchannels,respectively.Thismicrofluidicdevice
isnowreadytouse.
CompleteMicrofluidicDevice
Thediagramshowsthecompletemicrofluidicgelelectrophoreticdeviceafterfabrication.Thedevice
containspolyacrylamidegelswiththreedifferentporesizes.Thegelwithlargestporesizeisusedin
theloadingchannelstofacilitatetheelectrophoresisofthesampleandreagentsbypreventingbulk
flow.Thegelwithintermediateporesizeisusedintheseparationchannelforproteinseparation.
Finally,thegelwithsmallestporesizeisusedasasizeexclusionmembranefortheenrichment
process.ThedetaildescriptionsabouttheirfunctionswillbecoveredinthesectionentitledPrinciple
ofOperation.
Inthedevicedesign,alltheloadingchannelsareconnectedtotheloadingareas,whichareholes
drilledatthebeginning(beforeanodicbonding).Thesampleandreagentsareloadedintothere.In Completemicrofluidicgel
addition,someholesareusedasreservoirsforwastecollection.Thesearetheplacestowhichthe electrophoreticdevice [3]
electriccurrentflows.Itisworthnotingthatinadditiontobeingtheloadingareas,theyarealsoused
astheinsertionpointsforelectrodes,whichareconnectedtoaprogrammablesupplyvoltagesource.
Whennotinuse,thismicrofluidicgelelectrophoresisdevicemustkeepsubmerginginthebufferandstoreat4C.
II.PrincipleofOperation
Basedonthesameprincipleofelectrokinetictransportofchargedmolecules,themicrofluidicgelelectrophoresisworkssimilarlytothe
regularslabgelelectrophoresis,butwithmuchfasterprocessing,moresensitivedetection,andhighlyautomatedintegratedsystems.In
thissection,thefundamentaloperationofthemicrofluidicgelelectrophoresiswillbediscussedusingthemicrofluidicstructuredescribed
intheprevioussection.
Themicrofluidicgelelectrophoreticsystemtobediscussedconsistsofastructurecontainingmicrofluidicchannels/reservoirs,power
supply,andfluorescencedetectionsystem.Thechannelsandreservoirsusedtotransporttheanalytearedesignedsuchthattheyarefilled
withlargeporesizepolyacrylamidegel.Thislargeporesizegelfacilitatestheelectrokinetictransportoftheanalytebypreventingthe
bulkflow.[3]Thepowersupplyisdesignedsothatitisprogrammable.Apairofelectrodesandtheirpolaritiescanbeassigned
instantaneously.Thisprovidesamuchbettercontrolovertheelectrokineticprocessonlyattainableinmicrofluidicenvironment.Also
integratedintothesystemisthefluorescencedetectioncapability.Itisusedtodetecttheanalyteofinterestandquantifyitsconcentration.
Inthissection,astepbystepoperationprocedureincludingtheprinciplebehindeachstepwillbediscussed,assumingthatthetarget
analytesarenegativelycharged.Notethatthediscussionwillmainlyfocusontheimmunoassayapplication,whichismoregeneralizedto
thestudybyHerretal.[3]Alittlemodificationcanbemadetothesystemtobeusedinotherapplications.
LoadingaReporterofKnownConcentration
Asatoolforimmunoassaystudy,areporterspecifictoaparticularproteinofinterestisused.Actinglikeareceptorwithhighaffinityfor
atargetprotein,anantibodycanbeusedasthereporterforthedetectionofproteinorantigenbeinginvestigated.Withoutlossof
generality,thetermreporterwillbeusedthroughoutthissection.
Tobeginusingthedevice,themicrofluidicchannelsneedtobefilledwithbuffersolution.Then,thereportersolutionofknown
concentrationtogetherwithsamplesolutionisloadedintothedesignatedreservoir.Typicallyforthemicrofluidicdevice,thevolume
requiredperanalysisisroughlyintheorderofafewtensofmicroliters.Forthepurposeofdetectionandquantification,thereporteris
usuallylabeledwithfluorescencetag.Thedetectionandquantificationmethodswillbediscussedindetaillater.
Afterthefluorescentlylabeledreporterisloaded,apairofelectrodesconnectedtothereporterand
samplewastereservoirsareactivatedasshowninthediagram.Theelectricpotentialisthenapplied
acrossbothelectrodes.Asthereportermoleculesbecomechargedinthebuffer,theyare
electrokineticallymovedtowardthesamplewastereservoirasindicatedbytheredarrows.The
reportermolecules,however,areblockedbythesizeexclusionmembrane,whichallowsonly
particlesofsizelessthan10kDatopassthrough.Inthisloadingstep,onlytheionicbuffercan
penetratethemembrane.Attheendofthisfirstelectrophoresis,theincreasingnumberofthe
fluorescentlylabeledreportermoleculesgathersatthemembraneonthegelsideasshowninthe
inset.
LoadingaSampleContainingAnalyteofInterest
Electrokineticallyloading
Thenextprocessistoloadthesampleelectrokineticallytocombinewiththereporter,whichalready fluorescentlylabeledreporter [3]
gatheredatthemembrane.Likethereporter,theanalytemoleculesbecomechargedinthebuffer.To
begintheelectrophoresis,theelectrodecontactedtothereporterreservoirisdeactivated.The
electrodecontactedtothesamplereservoirisswitchedoninstead.Thepotentialacrossthesetwo
electrodescausesthechargedmoleculesofproteinsinthesampletomoveelectrokineticallytoward
thesamplewastereservoirasindicatedbyredarrows.
Oncethechargedmoleculesoftheanalytearrivethemembrane,someofthemwhosesizelessthan
10kDapassthroughthemembraneandarecollectedinthesamplewastereservoir.Onlythelarger
moleculesincludingtheproteinofintereststayonthegelsizeofthemembraneasshownintheinset.
Thisprocesshelpsincreasetheconcentrationoftheanalyte,improvingtheprobabilityofbinding
betweenthereporterandtargetprotein.However,thenontargetproteinsbecomemoreconcentrated
aswellandmightcauseareductioninsignaltonoiseratiolevel.Usingthereporterofhighbinding
specificitycanalleviatethisproblem.Itisworthnotingthatthesensitivityanddynamicrangeofthe
microfluidicgelelectrophoreticimmunoassaydependonthisenrichmentprocess.
Itneedstobedesignedcarefullysothattheelectrophoresisoccurslongenoughsothatmorereporters Electrokineticallyloadinga
andthetargetproteinsareboundtogether.Normally,theconcentrationofthereporterusedismuch sample [3]
higherthanthatofthetargetprotein.Asaresult,allthetargetproteinmoleculeswouldbemore
likelytobedetectedbythereportermolecules.Attheendofthissecondelectrophoresis,thereporter
anditscomplexincludingtheremainingnontargetproteinsstayonthegelsideofthemembrane.
AvoidRunningElectricCurrentThroughMembrane
DuringSeparation
Ithasbeenreportedthatthegelelectrophoreticseparation
experiencedirreproducibleresultswhenrunningtheelectric
currentthroughthemembrane.[3]Therefore,itisnecessaryto
avoidapplyingtheelectricpotentialacrossthemembrane
especiallyduringtheseparationprocess.Onepossible
solutionistoprogramthepowersupplysuchthatthe
electrodesareswitchedbeforethetargetanalytesenterthe
separationgel.Thediagramshowstheswitchingprocessthat
satisfiesthisrequirement.
Switchingelectrodestoavoidrunningelectriccurrentthroughthemembrane
Accordingtothediagram,theelectricpotentialisinitially duringelectrophoreticseparation [3]
appliedacrossthemembranejusttotransporttheanalytes
awayfromthemembrane.Beforetheanalytesenterthe
separationgel,theelectrodeattheupperleftbufferreservoirisdeactivated.Atthesametime,anewelectrodewiththesamepolarityat
theupperrightbufferreservoiristurnedon.Itcanbeseenthattheflowofnegativelychargedanalytesremainsinthesamecoursetoward
thebufferwastereservoir.
Itisworthnotingthatthereareothercombinationsofelectrodesthatcanbeusedtoperformthisstepaswell.Theconceptissimplynot
toallowelectrophoresistooccuracrossthemembraneduringtheseparationstep.Attheendofthethirdelectrophoresis,allcharged
moleculesarereadyforthegelelectrophoreticseparation.
GelSeparation
Inthisstep,thereportermoleculesandtheircomplexareseparatedbymeansofgelelectrophoresis.Theprocesscontinuesby
electrokineticallycarryingthechargedmoleculesintotheseparationgel.Sincethereportermoleculesandtheircomplexhavesimilar
chargetomassratio,theseparationbetweenthesetwospeciesisbasedonlyonthedifferenceintheirmolecularweights(MWs).Itis
worthnotingthatallnontargetproteinsarenotinvestigatedinthisimmunoassay.Onlythereporter
moleculesandtheircomplexareunderexamination.
Sincethereportermoleculesarelabeledwithfluorescentdye,asinglepointlasercanbeusedto
inducethefluorescence.Thislaserinducedfluorescence(LIF)ismonitoredbyadetectorthatwill
detectthepresenceoftheunboundreporteranditscomplex,andthenrelatetheintensityofthe
detectedfluorescencetotheconcentrationofbothspecies.Thisfluorescencedetectionsystemis
placedacrosstheseparationchannelnearthebufferwastereservoir.Theconceptsofdetectionand
quantificationaredescribednext.
FluorescenceDetectionandResults
Electrophoreticseparationof
Thesinglepointlaserinducedfluorescence(LIF)isameanusedtodetectandquantifythereporter unboundreporteranditscomplex
moleculesandtheircomplex.Whenthemoleculesacrossthelaserbeam,theattacheddyesemit detectablebylaserinduced
fluorescencewhoseintensityismeasuredbythedetector.Thedetectorthengeneratesthe fluorescence [3]
electropherogramcorrespondingtothemeasuredintensity.Theareaundertheelectropherogrampeak
canberelatedtotheconcentrationofthetargetprotein.
Theunboundreportercanbedistinguishedfromthecomplexbasedonthefactthattheunbound
reportermoleculehaslowermolecularweight(MW)thanitscomplex.Therefore,theunbound
reportermoleculestravelelectrokineticallyfasterintheseparationgelandaredetectedfirst.The
correspondingintensitygivesthefirstpeakintheelectropherogram.
Fluorescentdetection,
electropherogram,andgellike
Incaseofasinglepairofligandandreceptor,therewillnormallybetwopeaksinthe
electropherogram.UsuallythefirstpeakbelongstothereceptororreporterofsmallerMW,followed plots [3]
bythesecondpeakcorrespondingtotheligandreceptorcomplexoflargerMW.Thisinformationcan
alsoberepresentedbygellikeplots,whicharecomputergenerated.Inthegellikeplots,thetopmostbandrepresentstheunbound
reportermoleculesthatarrivefirst.Thebrightnessofthebandconveystheinformationaboutthedegreeoffluorescenceintensity
detected.Thewidthofthebandcorrespondstothewidthofthepeak,whichconveystheinformationaboutthemigratingtimeofthe
analyte.
Itisworthmentioningthattheprobabilityofbindingbetweenbothreceptorandligandisasignificantfactortothesensitivityand
accuracyoftheimmunoassay.Therefore,theenrichmentprocessplaysanimportantroleinraisingthisprobability.Toenhancethis
enrichmentprocess,eitherpreprocessingthesample(offchip)orincreasingtheperiodoftheelectrophoreticsampleloadingisprovento
beuseful.
III.ClinicalApplications
Oneofclinicalapplicationsreportedwastheuseofthismicrofluidicgelelectrophoreticdeviceinassistingoraldiagnosis.[1][3]Itwas
usedforearlydiagnosisoftheperiodontaldiseaseandtomonitorthediseasestateanditsdevelopmentfromhumansaliva.Periodontal
diseaseorperiodontitisisaputativeoraldiseasethatdestroyscollagenandcausesamajortissuedamage,connectivetissueattachment
loss,andboneloss.Foundintheperiodontitispatientsalivawerematrixmetalloproteinase8(MMP8(http://www.rcsb.org/pdb/explore/e
xplore.do?structureId=2OY4)),interleukin1beta(IL1B),andCtelopeptidepyridinolinecrosslinks(ICTP).Theseproteinswere
identifiedasdiseasebiomarkersandbecamethetargetproteinsfordetectionandmonitoringthisoraldisease.[1]Herretal.demonstrated
thedetectionandquantificationofMMP8usingthismicrofluidicgelelectrophoreticdevice.
Intheirstudy,themonoclonalantibody(http://en.wikipedia.org/wiki/Monoclonal_antibodies)forMMP8wasusedasareporter,which
hadthespecificityforbindingonlytotheMMP8inthesaliva.Theuseofthismonoclonalantibody( MMP8)hadanadvantageinthat
iteliminatedtheneedforamatchedpairantigenantibody.Inthereportermixture,thebovineserumalbumin(BSA)proteinstandardwas
alsoaddedasareference.Both MMP8*andBSA*werefluorescentlylabeledforthedetectionpurpose.Notethattheasteriskisused
todenotethefluorescentlylabeledanalyte.Accordingtotheirstudy,1nMof MMP8*and1nMofBSA*wereusedinthemixture.
Inthemicrofluidicdevice, MMP8*boundonlytoMMP8biomarkerformingacomplexofsimilarchargetomassratio.The
unboundedantibodyanditscomplexwereseparatedfromeachotherbytheseparationgelbasedonthedifferenceintheirsizesand
detectedindividuallyasdescribedinthePrincipleofOperationformicrofluidicgelelectrophoresis.Inthisimmunoassayapplication,the
BSA*withthehighestmobilitywasdetectedfirst,followedbytheremaining MMP8*andMMP8complex,respectively.The
correspondingpeakswereshownintheelectropherogramandgellikeplots.Thepeakareaorthewidthofthebandingellikeplotwas
usedtocalculatetheconcentrationofMMP8.
TobeabletoquantifytheconcentrationoftheendogenousMMP8inthesalivasamplecollectedfromthepatients,acalibrationcurve
wasrequiredtobegeneratedfirst.ThecalibrationcurvewasobtainedfromtheanalysesofknownMMP8concentrations.Aseriesof
experimentswereperformedbyaddingknownconcentrationsoftherecombinantMMP8inthedilutedsalivafromhealthypatientsand
thenrunningthemicrofluidicgelelectrophoresis.Fromtheelectropherogram,thepeakareasoftheMMP8complexwerenormalized
withthepeakareaofBSA*andthenplottedagainstthecorrespondingconcentrations.Anonlinearleastsquaresfittingmethodusinga
fourparameterlogisticmodel[3]wasusedtoobtainedthecalibrationcurve.Basedonthiscalibrationcurve,theconcentrationofthe
endogenousMMP8complexwaspredictedfromitsnormalizedpeakarea.ItwasreportedthattheaverageconcentrationsoftheMMP8
inthehealthyanddiseasesubjectswere64.6+/16.4ng/mLand623.8+/204.0ng/mL,respectively.[3]Itisworthnotingthatthe
concentrationofMMP8inthepatientsclassifiedasperiodontallydiseasedexhibitedthedynamicactivityofthedisease.
Thevalidityoftheresultsobtainedfromthemicrofluidicgelelectrophoresiswasverifiedbycomparingwiththoseobtainedfromthe
conventionalenzymelinkedimmunosorbentassay(ELISA(http://en.wikipedia.org/wiki/ELISA)).AsreportedbyHerretal.,theresults
obtainedfromthemicrofluidicdevicewerehighlycorrelatedwiththosefromELISAwithr2=0.979,whererisPearsonproductmoment
correlationcoefficient.Thismicrofluidicbasedimmunoassay,however,hadseveraladvantagesovertheconventionalcounterpartinthat
itbypassedthetimeconsumingreactionandwashingstepsrequiredbyELISA,wasmoreautomated,requiredonlysingleantibody
makingitapplicableforwiderrangeofapplications,andneededmuchlessamountofsalivasampleperanalysis.Inaddition,itdidnot
requiresurfaceimmobilizationoftheantibody.
InadditiontomeasuringtheconcentrationofMMP8intheperiodontitispatients,aclinicalexaminationwasalsoperformedtocorrelate
theanalyticaldatawithphysiologicalsymptoms.ItwasfoundthatMMP8washighlycorrelatedwithboneandtissueloss,buthavingno
correlationwithbleedinguponprobing.WithhighlysensitivedetectionofMMP8biomarker,thismicrofluidicbasedimmunoassay
couldprovideearlydiagnosisthatwouldimprovetheclinicaltreatmentofthisdisease.Furthermore,withtheuseofMMP8inhibitorto
reducecollagendegradation,itwaspromisingthatthismicrofluidicgelelectrophoreticdevicecouldbeusedtomonitorandtrackthe
progressofMMP8inhibitortherapyaswell.
MicrofluidicCapillaryElectrophoresis
Inthissection,anothertypeofmicrofluidicelectrophoresiswillbedescribed.Unlikemicrofluidicgelelectrophoresis,microfluidic
capillaryelectrophoresisoperatesontheprincipleofelectrokineticofbulkflow.Thedifferenceinchargetomassratioofanalytesisthe
fundamentalofelectrophoreticseparationincapillarymicrofluidicchannel.Withouttheneedofsievingmedium,themicrofluidic
capillaryelectrophoresisbecomesmorefavorablethanitsgelcounterpartintermsoftheeaseinfabricationandreusability.Thedetail
fabricationprocessandprincipleofoperationincludingclinicalapplicationsofthisdevicearedescribedasfollows.Notethatthe
fabricationprocessandprincipleofoperationexplainedbelowarebasedonthepublishedworkbyBackofenetal.2002.[10]
I.FabricationProcess
ThefabricationofmicrofluidicstructureusingPDMSiseasierandmuchlessexpensivethanusingglass.Theprocessbeginswith
creatingamasterformoldingthePDMS,whichcanbeobtainedbypatterningaphotoresist.Afterdeveloping,amixtureofPDMSand
crosslinkerispouredoverthepatternedphotoresistandthencured.ThePDMSispeeledoutandcuttocreateanaccesstothereservoir.
Inthefinalstep,thispatternedPDMSisattachedpermanentlytoaglassbaseandreadytouse.Thedetailstepbystepprocedureis
describedbelow.
CreatingaMasterforPDMSMolding
Materials:[10]
1.Photomask
2.4inchsiliconwafer
3.SU850negativephotoresist
4.Propyleneglycolmethyletheracetatedeveloper Diagramdescribingafabrication
processtocreateamasterfor
Beginningwithapreparationprocess,aphotomaskiscreatedfirst.Thephotomaskusedisusuallya PDMSmoldingusingnegative
contactphotomaskthatcanbemadeofglassorsimplyatransparencywiththedesignedmicrofluidic photoresistandstandard
structurallayoutprintedonit.Thephotomaskisusedtotransferthismicrofluidicpatternontoa photolithography [10]
photoresist,whichisspincoatedontoasiliconwafer.Itisworthnotingthatthechoiceofphotoresist
tobeusedaffectsthedesignofphotomask.Forexample,ifusingthenegativephotoresist,allthe
microfluidicpatternsneedtobedesignedusingclearfieldsothattheUVlightcangothroughandpolymerizethecontactareasofthe
negativephotoresist.Theunexposedareaswillbewashedawayinadeveloper.Ontheotherhand,thedesignedpatternsarethedarkfield
ofphotomaskforthepositivephotoresist.
Beforecoatingthephotoresist,thesiliconwaferisneededtobecleanedfirst.AstandardRCAcleaningprocessisapplicable.Then,the
negativephotoresistisspincoatedonthesiliconwaferandprebaked.ExposingundertheUVlight,thecoatednegativephotoresistis
patternedwiththedesignedmicrofluidiclayoutfromthephotomask.Theexposedphotoresististhenpostbakedanddeveloped.Asa
result,onlytheexposedareasofthenegativephotoresistremainasshowninthediagram.
MoldingPDMStoFabricateMicrofluidicStructure
Materials:[10]
1.PDMSoligomer
2.Sylgard184crosslinker
AfterthemasterforPDMSmoldingisready,amixtureofPDMSandcrosslinkerisprepared.A DiagramshowingthePDMS
moldingprocessusingthe
10:1ratioofthepolymerandcrosslinkerisapplicable.[10]Thedegassedmixtureisthenpouredonto developed,patternedphotoresist
thewafertocovertheentirephotoresistmaster.Finally,themoldedPDMSiscuredintheovenat70 [10]
Cfor1hour.[10]Thisprocessstepissummarizedinthediagram.Inaddition,acrosssectionalview
ofthePDMSmoldedonthewaferisalsoprovided.
Aftercuring,thePDMSispeeledoutofthewafer.ThePDMSwiththetransferredmicrofluidicstructureisfurtherprocessedbycreating
anopeningforeachcircularreservoirregion.Thisopeningwillbeusedtoloadsampleorreagentandaplacetoapplyvacuumand
pressure,whichisregulatedbyasyringe.
FinalizeMicrofluidicCapillaryElectrophoreticDevice
Materials:
1.Glassplate
2.Needleprobe
Aglassplatewithcoatedpatternedelectrodesisusedtoenclosethemicrofluidicchannelsand
reservoirs,andatthesametime,provideselectricalconnectionstothefluidinreservoirs.The
electrodescanbefabricatedonglasssubstratebymeansofevaporationofchromeandplatinum. Diagramshowingthecomplete
Chromeisusedasanadhesivelayerbetweenglasssubstrateandplatinumelectrodes.Thepatterning microfluidiccapillary
ofthemetalscanbeachievedbyaregularphotolithography,whichrequiresanotherphotomask(a
electrophoreticdevice [10]
transparency),toformtheelectrodesonglass.Itisworthnotingthatacombinationofpatterned
electrodesandneedleprobeisusedinthefinalmicrofluidiccapillaryelectrophoreticsystemas
showninthediagram.Theplatinumneedleprobeisusedtosupplyhighelectricpotentialforelectrophoreticpurposeandsimultaneously
asanelectrochemicalsensor.
ThePDMSispermanentlyattachedtotheglassplatebyoxidationusingplasma.Acovalentbondingbetweenoxygenandsiliconatoms
(OSiO)providesapermanentsealbetweenbothmaterials.Thecompletemicrofluidiccapillaryelectrophoreticdeviceisshowninthe
diagram.
II.PrincipleofOperation
Ingeneral,theoperationofmicrofluidiccapillaryelectrophoreticdevicecanbedescribedbythreefundamentalstepsloading
sample/reagents,formingsampleplug,andelectrophoreticseparation.Theloadingprocessdescribedhereinvolvesmanualloadingusing
syringestoregulatetheflow.Afterallsampleandregentsareloaded,asampleplugisformed.Thisstepneedstobedesignedcarefully
sincetheconcentrationoftheanalytesreliesonthisstep.Inthelaststep,theanalytesareseparatedelectrophoretically.Unlikethesize
basedseparationinthe(microfluidic)gelelectrophoresis,theseparationby(microfluidic)capillaryelectrophoresisisbasedonthe
differencesinchargetomassratiosoftheanalytes.
Itisworthnotingthatthemicrofluidiccapillaryelectrophoreticsystembeingdiscussedhereisusedasananalyticalplatformwhereall
thesamplepreprocessingstepsareperformedoffchip.Thefollowingdescriptionfocusesmainlyontheanalysisofnegativelycharged
analyteinthesample.NotethattheprincipleofoperationdescribedbelowisbasedinpartonthepublishedworkbyBackofenetal.
2002.[10]
LoadingProcess
Thesystemsetupbeginswithloadingthemicrofluidicreservoirswithabufferandpreprocessedsamplesolutionsuchthatallthe
reservoirsexceptthesamplereservoircontainthebuffer.Continuingwithfillingthemicrofluidicchannelswithbuffer,syringesare
connectedtothetopopeningsofthereservoirsandusedtoregulatetheflow.Vacuumandpressureareapplieduntilthechannelsare
filledwiththebuffer.
Thenextprocessistoequilibratethesampleandbufferbymeansofelectrophoresis.Todoso,thehighvoltagesourcesareconnectedto
thesystemasshowninthediagram.OnepossibleconfigurationistheuseofmultiplesupplyunitssuchastwounitsofU1=0.5kVanda
singleunitofU2=1.2kV.Thisprocessallowsthebufferandsampletolocalizeinthechannelsasshowninthediagram.
FormingSamplePlug
Forthesampletobeanalyzed,apredefinedandcontrollableportionofthesampleneedstobe
injectedintotheseparationchannel.Thispredefinedandcontrollableportionofthesampleis
referredtoasasampleplug.Accordingtothemicrofluidicstructurebeingdiscussed,thesampleplug
isformedbyahydrodynamicflowofthesamplesolutionduetothedifferenceinthesolutionlevels
inthereservoirs.Thishydrodynamicflowonlyoccurswhenallthehighvoltagesourcesareswitched
Allmicrofluidicchannelsfilled
off.
manuallyandequilibratedby
Asdepictedbythediagram,theportionofsamplesolutionflowsthroughthesmallconnecting electrophoresis [10]
channelintothechannelsleadingtothebufferandbufferwastereservoirs.Thissampleportioncan
bedividedintotwoportsindicatedbyblueandyellowdashedoutlinesasshownintheinset.The
blueoutlineisthesampleplugwhosevolumecanbespecifiedbydesign.Theyellowdashedoutline
indicatestheportionofthesamplethatwillflowbacktowardthesamplewastereservoirwhen
switchingthehighvoltagesourcesbackon.
ElectrophoreticSeparation
Whenswitchingonallthehighvoltagesources,thenegativelychargedanalytemoleculesandbuffer Samplesolutionflowingthrough
ionsstarttoflowagainbyelectrophoresis.Atthesmallconnectingchannel,theportionofsample thesmallconnectingchanneland
solutionbeginstosplitintotwopartsandthenmoveawayintheoppositedirections.Asdepictedin enteringthechannelsleadingto
theinset,thepartofsamplesolutionindicatedbybluedashedoutline,orthesampleplug, thebufferandbufferwaste
electrokineticallymovesalongtheseparationchanneltowardthebufferwastereservoir.Ontheother reservoirs [10]
hand,thepartofsamplesolutionoutlinedbytheyellowdashedlineelectrokineticallymoveaway
towardthesamplewastereservoir.Thesmallconnectingchannelisthenfilledbythebufferas
occurredoriginally.
Intheseparationchannel,thenegativelychargeanalytesinthesampleplugareseparatedduetothe
differencesintheirchargetomassratios.Eachanalyteisdetectedbytheelectrochemicalsensor
placedattheendbufferwastereservoir.Fortheelectrochemicalsensorusedinthismicrofluidic
capillaryelectrophoreticsystem,thedetectionofeachanalyteisbasedonareductioninvoltagedrop
acrosstheneedleprobeandreferenceelectrode.Thesevoltagedropscanbeplottedinthe
electropherogramandcanbeusedtostudytheanalytecomponentssuchasproteinsorpeptidesinthe
Sampleplugelectrokinetically
samplesolution.
movingalongtheseparation
channeltowardthebufferwaste
reservoir [10]
III.ClinicalApplications
ThedetectionandquantificationofbiomarkersinpatientswithskinlesionsreportedbyGuzmanetal.2008[5]isoneoftheclinical
applicationsforthemicrofluidiccapillaryelectrophoresis.Basedonthesamefundamentalconceptdescribedpreviously,amore
sophisticateddesignofmicrofluidiccapillaryelectrophoresissocalledtheimmunoaffinitycapillaryelectrophoresis(IACE)wasusedin
theirstudy.IACEisalabonachipthatutilizesthemicrofluidictechnologytoincorporatetheaffinitybasedpurification,enrichment,
andelectrophoreticseparationprocessesinonesinglemicrochip.
Intheclinicalstudyofbiomarkersforthisinflammatorydisease,IACEwasusedtoanalyzethemicrodissectedsamplesfromthe
patientswithdifferentstagesofskindamage.Twelvedifferentantibodieswereusedtocapturetwelvecorrespondingtarget
proteins/peptidesbeingconsideredasbiomarkers.Thisaffinitybindingalsoassistedtheisolationoftargetanalytesfromnontarget
analytesinmicrofluidicenvironment,thusimprovingthesubsequentenrichmentprocessandreducingbackgroundnoiseduringthe
analysis.Asreported,theintegratedenrichmentfeatureincreasedthesensitivityandenhancedthelowdetectionlimitofIACE.The
concentrationaslowasafewnanogrampermillilitercouldbedetectedandquantified.Withthishighlysensitivecapability,itwasalso
reportedthatIACEcouldbeusedtomonitortheprogressivepatternsofthedisease.TheresultsobtainedbyIACEwereprovenconsistent
withthegoldstandardlikethetraditionalhistopathology.
Notes
1.HerrAE,HatchAV,GiannobileWV,ThrockmortonDJ,TranHM,BrennanJS,SinghAK."Integratedmicrofluidicplatformfor
oraldiagnostics"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2572166)AnnNYAcadSci.Authormanuscript
availableinPMC(2008).
2.XuX,LiL,WeberSG."Electrochemicalandopticaldetectorsforcapillaryandchipseparations"(http://www.pubmedcentral.nih.g
ov/articlerender.fcgi?artid=1832263)TrendsAnalytChem.AuthormanuscriptavailableinPMC(2008).
3.HerrAE,HatchAV,ThrockmortonDJ,TranHM,BrennanJS,GiannobileWV,SinghAK."Microfluidicimmunoassaysasrapid
salivabasedclinicaldiagnostics"(http://dx.doi.org/10.1073/pnas.0607254104)ProcofNatAcadSci.104(13):52685273(2007).
4.BarryR,IvanovD."Microfluidicsinbiotechnology"(http://dx.doi.org/10.1186/1477315522)JNanobiotechnology2:2(2004).
5.GuzmanNA,BlancT,PhillipsTM."Immunoaffinitycapillaryelectrophoresisasapowerfulstrategyforthequantificationoflow
abundancebiomarkers,drugs,andmetabolitesinbiologicalmatrices"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=
2659498)Electrophoresis.AuthormanuscriptavailableinPMC(2009).
6.GongM,WehmeyerKR,LimbachPA,AriasF,HeinemanWR."Onlinesamplepreconcentrationusingfieldamplifiedstacking
injectioninmicrochipcapillaryelectrophoresis"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2442013)AnalChem.
AuthormanuscriptavailableinPMC(2008).
7.GongM,WehmeyerKR,StalcupAM,LimbachPA,HeinemanWR."Studyofinjectionbiasinasimplehydrodynamicinjectionin
microchipcapillaryelectrophoresis"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2442562)Electrophoresis.Author
manuscriptavailableinPMC(2008).
8.HsiehJF,ChenST."Comparativestudiesontheanalysisofglycoproteinsandlipopolysaccharidesbythegelbasedmicrochip
andSDSPAGE"(http://dx.doi.org/10.1063/1.2399892)Biomicrofluidics1(2007).
9.WakoChemicals(http://www.wakousa.com/specialty/specialty_focus.html)
10.BackofenU,MatysikFM,LunteCE."Achipbasedelectrophoresissystemwithelectrochemicaldetectionandhydrodynamic
injection"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2519826)AnalChem.AuthormanuscriptavailableinPMC
(2008).
References
HerrAE,HatchAV,GiannobileWV,ThrockmortonDJ,TranHM,BrennanJS,SinghAK."Integratedmicrofluidicplatformfor
oraldiagnostics"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2572166)AnnNYAcadSci.Authormanuscript
availableinPMC(2008).
XuX,LiL,WeberSG."Electrochemicalandopticaldetectorsforcapillaryandchipseparations"(http://www.pubmedcentral.nih.g
ov/articlerender.fcgi?artid=1832263)TrendsAnalytChem.AuthormanuscriptavailableinPMC(2008).
HerrAE,HatchAV,ThrockmortonDJ,TranHM,BrennanJS,GiannobileWV,SinghAK."Microfluidicimmunoassaysasrapid
salivabasedclinicaldiagnostics"(http://dx.doi.org/10.1073/pnas.0607254104)ProcofNatAcadSci.104(13):52685273(2007).
BarryR,IvanovD."Microfluidicsinbiotechnology"(http://dx.doi.org/10.1186/1477315522)JNanobiotechnology2:2(2004).
GuzmanNA,BlancT,PhillipsTM."Immunoaffinitycapillaryelectrophoresisasapowerfulstrategyforthequantificationoflow
abundancebiomarkers,drugs,andmetabolitesinbiologicalmatrices"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=
2659498)Electrophoresis.AuthormanuscriptavailableinPMC(2009).
GongM,WehmeyerKR,LimbachPA,AriasF,HeinemanWR."Onlinesamplepreconcentrationusingfieldamplifiedstacking
injectioninmicrochipcapillaryelectrophoresis"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2442013)AnalChem.
AuthormanuscriptavailableinPMC(2008).
GongM,WehmeyerKR,StalcupAM,LimbachPA,HeinemanWR."Studyofinjectionbiasinasimplehydrodynamicinjectionin
microchipcapillaryelectrophoresis"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2442562)Electrophoresis.Author
manuscriptavailableinPMC(2008).
HsiehJF,ChenST."Comparativestudiesontheanalysisofglycoproteinsandlipopolysaccharidesbythegelbasedmicrochip
andSDSPAGE"(http://dx.doi.org/10.1063/1.2399892)Biomicrofluidics1(2007).
WakoChemicals(http://www.wakousa.com/specialty/specialty_focus.html)
BackofenU,MatysikFM,LunteCE."Achipbasedelectrophoresissystemwithelectrochemicaldetectionandhydrodynamic
injection"(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2519826)AnalChem.AuthormanuscriptavailableinPMC
(2008).
ProteinSeparationsCentrifugation
Presentation
Printversion
IntroductiontoCentrifugation
Centrifugationisoneofthemostimportantandwidelyappliedresearchtechniquesinbiochemistry,cellularandmolecularbiology,and
inmedicine.Inthefieldofproteomicsitplaysavitalroleinthefundamentalandnecessaryprocessofisolatingproteins.Thisprocess
beginswithintactcellsortissues.Beforetheproteinscanbeobtained,thecellsmustbebrokenopenbyprocessessuchassnapfreezing,
sonication,homogenizationbyhighpressure,orgrindingwithliquidnitrogen.Oncethecellshavebeenopenedupalloftheircontents
includingcellmembranes,RNA,DNA,andorganelleswillbemixedinthesolventwiththeproteins.
Centrifugationisprobablythemostcommonlyusedmethodforseparatingoutallthenonproteinmaterial.
Withinthecentrifugesamplesarespunathighspeedsandtheresultingforcecausesparticlestoseparate
basedontheirdensity.
UsesofCentrifugation
Centrifugationiscapableof:
Removingcellsorothersuspendedparticlesfromtheirsurroundingmilieuoneitherabatchora
continousflowbasis
Tabletopcentrifuge
Separatingonecelltypefromanother
Isolatingvirusesandmacromolecules,includingDNA,RNA,proteins,andlipidsorestablishingphysicalparametersofthese
particlesfromtheirobservedbehaviorduringcentrifugation
Separatingfromdispersedtissuethevarioussubcellularorganellesincludingnuclei,mitochondria,cholorplasts,golgibodies,
lysosomes,peroxisomes,glyoxysomes,plasmamembranes,endoplasmicreticulum,polysomes,andribosomalsubunits.
Oncethemixtureofproteinshasbeenisolatedusingcentrifugationthescientististhenabletouseoneofseveralmethodstoseparateout
individualproteinsforfurtherstudy.Formoreinformationonproteinpurification/separationseeProteinSeparationsChromatography
andProteinSeparationsElectrophoresis.
Nextsection:HistoryoftheCentrifuge
References
SubscriptionBasedReferences
1.Sheeler,P."CentrifugationinBiologyandMedicalScience."DeptofBiology,CaliforniaStateUniversity,Northridge,California
OpenAccessReferences
1.Claude,A.&Potter,J.S."IsolationofChromatinThreadsFromTheReastingNucleusofLeukemicCells"(http://www.jem.org/cg
i/reprint/77/4/345.pdf)TheJournalofExperimentalMedicine.
EmergingandMiscellaneousProteomicsTechnologies
Presentation
Printversion
EmergingandMiscellaneousTechnologiesinProteomics
Thispartofthebookwillbeanareawhereproteomicstechniquesthathavebeennewlydevelopedwillbediscussed.Techniquesthatdo
notcurrentlyfitintoanyotherpartofthebookcanalsobeaddedtothispage.Aschaptersorsectionsareaddedelsewherethatdiscuss
thesetechniquesinthecontextofagreaterproteomicsproblem,theinformationonthispagewillbemovedtothosepages.
XRayTomography
DescriptionandDiscussionofXrayTomography.
AnewbranchofXraymicroscopyisbeingusedinproteomicsanalysis.ThisiscalledXrayTomography.Thismethodusesprojected
imagestocalculateandreconstructa3Dobject.Thistechnologyisbeingusedinproteomicstodeterminethelocationoflabeledproteins
orlargecomplexeswithinacell.Thistechniquecanalsobeusedinconjunctionwithimagesofcellsfromlightbasedmicroscopesto
helpidentifywhereaproteinislocatedandhowthislocationfactorsintoitsfunctionandidentification.
IntroductiontoProteoinformatics
Proteoinformaticsistheuseofbioinformaticsandcomputationalbiologytechniquessolelywithintherealmofproteinidentificationand
proteomics.Proteoinformaticsiscurrentlyinitsinfancyandthelargestworkbeingdoneisonstandardizingdatabasesanddata
submission.Otherproteoinformaticworkisbeingdoneontheimageanalysisof2Dgelsandotherimagesinproteomicsusedtohelp
identifyandannotateproteinsintheproteome.
ProteinIdentificationDatabase
WhatareProteinIdentificationDatabases?
ProteinIdentificationsDatabasessuchasProFoundatRockefellerUniversityandProteinProspectorattheUCSFMassSpectrometry
Facilityareusedtohelpidentifyproteinsfoundwithproteomicstechniquessuchasmassspectrometry.Digestionofproteinsintopeptide
fragmentsallowseachproteintobreakapartinadifferentway,resultinginauniquepeptidefingerprintthatcanbeusedtoidentifythe
protein.Themassesofthesefragmentsaswellasthemolecularweightsandisoelectricpointsarewhatisstoredinmanyofthese
databases.Thisdatacanbeusedtoperformhighthroughputproteinidentification.
TheFutureofProteinIdentificationDatabases
Inordertocontinueadvancingthecauseofmappingthehumanproteome,internationaldatabasesneedtobeestablishedwhichintegrate
bothtranscriptomeandproteomedata.TheHumanProteomeOrganizationiscurrentlyworkingonestablishingadefinedstandardfor
datasubmissionandannotationforthemanydifferentproteomicstechniquescurrentlyusedtoidentifyandannotateproteins.
NewTechniquesinImageAnalysis
AccordingtotheImageAnalysisWikipediapage,"Imageanalysisistheextractionofmeaningfulinformationfromimages."Intermsof
Proteomics,imageanalysiscanbeusedtocomparedifferentimagesgeneratedusingproteomicstechniques,suchas2DPAGEgel
images.Newprogramsarebeingdevelopedthatwillhelptooptomizeandautomatetheprocessoflocatingaproteinspotbetweentwo
gelimagesinordertoidentifythedifferencesbetween2DPAGEgels.Otherprogramscanbeusedtohelpcleanupandremove
variabilitybetweentheseimagesaswell.
LaserCaptureMicrodissection
LasercapturemicrodissectionorLCMisaprocessthatisolatesandremovesdistinctpopulations
fromatissue.Thiswillfacilitatethecomparisonofdiseasedtissuewithnormaltissuefroman
organism.
InLCM,aninfraredlaserbeammeltsathermosensitivepolymerfilmthattrapsaspecificgroup
ofcells.Thispolymerfilmisthenextractedandmovedtoatesttubewhereanextractionbufferis
usedtoremovethegroupsofcellsformoreadvancedproteomicsanalysissuchas2DPAGE,Ion
Chromatography,etc.Thistechnologywillbecomemoreusefulassystemswithhighersensitivity
foranalysisofsmalleramountsoftissuebecomedevelopedandrealized.
DiagramofLaserCapture
ProteomicComplexDetectionusingSedimentation Microdissection
ApproachessuchasTAPtagging,whichrequiretheadditionoffusionproteins,caninterferewith
proteininteractionsthatwouldhavenormallyoccurred.Manytimesittakesagreatdealofworktoexpressthesetaggedproteins,sothis
techniqueisusedtogiveevidencethatthereisastableproteincomplexdetectedearlyonintheproteomicsexperimentbeforemore
laboriousapproachesareusedtoisolateandidentifytheproteincomplexesofinterest.IssuesalsooccurinMSand2DGelprocesses
whereonecannotbesurethataportionofagelspotisthedesiredproteinbecausemultipleproteinscouldbetravelingtogetherinthat
spotasacomplex.
Thisiswhereproteomiccomplexdetectionusingsedimentation(ProCoDeS)isapplicable.ProCoDeSisatechniqueforthehigh
throughputidentificationofbothsolubleandmembraneproteinsthatarefoundinstablecomplexes.Relativesizesofproteincomplexes
areestimatedviatheirsedimentationinagradient.InthiscasearatezonalgradientorRZGisusedtobetterestimatetherelativesizeof
proteincomplexes.ThedistributionofaproteinofinterestinthissedimentationcanbedetectedusingclassictechniquessuchasWestern
BlottingornewertechniquessuchasICAT.Thiscanbedoneforalargenumberofproteins.Thus,ProCoDeScanbeusedtoidentify
stableproteincomplexes.ProCoDeSisespeciallywellsuitedforthescreeningofunrefinedcellularmaterialtohelpfindnewproteins
thatcannotbediscoveredbecausetheyexistinproteincomplexessuchasproteinsfoundinproteinmembranes.
NextChapter:ProteinIdentificationMassSpectrometry
References
1.Hartman,N.T.,etal."ProteomicComplexDetectionUsingSedimentation"(http://pubs.acs.org/cgibin/article.cgi/ancham/2007/7
9/i05/html/ac061959t.html)Anal.Chem.,79,5,20782083,2007.
2.NCTProteomicsGroup"EmergingTechnologies"(http://dir.niehs.nih.gov/proteomics/emerg5.htm)NationalInstitutesof
EnvironmentalHealthSciences
3.NCTProteomicsGroup"ProteoInformatics"(http://dir.niehs.nih.gov/proteomics/informtx.htm)NationalInstitutesof
EnvironmentalHealthSciences
4."Wikipedia:ImageAnalysis(http://en.wikipedia.org/wiki/Image_analysis)
5."Wikipedia:Proteomics(http://en.wikipedia.org/wiki/Proteomics)
6."Wikipedia:XrayTomography(http://en.wikipedia.org/wiki/Xray_Tomography)
ProteinIdentificationMassSpectrometry
Presentation
Printversion
ContactwikipediauserRiticeninjasregardingcontributions/corrections.
Introduction
MassSpectrometryOverview
Massspectrometryisatechniqueinwhichgasphasemoleculesareionizedandtheirmassto
chargeratioismeasuredbyobservingaccelerationdifferencesofionswhenanelectricfieldis
applied.Lighterionswillacceleratefasterandbedetectedfirst.Ifthemassismeasuredwith
precisionthenthecompositionofthemoleculecanbeidentified.Inthecaseofproteins,the
sequencecanbeidentified.MostsamplessubmittedtomassSpectrometryareamixtureof
compounds.Aspectrumisacquiredtogivethemasstochargeratioofallcompoundsinthe
sample.Massspectrometryisalsoknownas'massspec'orMSforshort.Massspectrometry
throwslightonmolecularmechanismswithincellularsystems.Itisusedforidentifyingproteins,
functionalinteractions,anditfurtherallowsfordeterminationofsubunits.Othermoleculesin
cellssuchaslipidcomponentscanalsobedefined.
Amassspectrometeriscomposedofseveraldifferentparts:asourcethationizesthesample,the
analyzerthatseparatestheionsbasedonmasstochargeratio,adetectorthat"sees"theions,and
adatasystemtoprocessandanalyzetheresults.Youcanalsomeasurerelativeabundanceofan
ionusingmassspectrometry.Differentcompoundshavedifferentialionizationcapabilitiesand
MALDITOFMS
thereforeintensityofyourionisnotadirectcorrelationtoconcentration.
Massspectrometrycanbeahighthroughputanalyticalmethodduetotheabilityforamassspectrumtobemeasuredrapidlyandwith
minimalsamplehandlingascomparedtogelmethods.
Itisananalyticalmethodwhichhasavarietyofusesoutsideofproteomics,suchasisotopeanddating,tracegasanalysis,atomic
locationmapping,pollutantdetection,andspaceexploration
HistoryofMassSpectrometry
Thehistoryofthistechniquefindsitsrootsinthefirststudiesofgasexcitationinachargedenvironment,morethan100yearsago.This
pioneeringworkledtotheidentificationoftwoisotopesofneon(neon20andneon22)viamasstochargerationdiscriminationbyJ.J
Thomsonin1913.Overthenextfiftyyearsthefundamentalbasisofthetechniquewasfurtherdeveloped.Afterthecouplingofgas
chromatographytoMassSpectroscopyin1959byresearchesatDowChemical,thefullpotentialofthetechniqueasahighlyaccurate,
quantitativemethodforexploringcompoundswasrealized,spurringawaveofdevelopmentswhichcontinuetothepresentday.The
precisionofmassspectrometryledtothediscoveryofisotopes.
ImplicationsofMassSpectrometryforProteomicsApplications
Thetechniqueofmassspectrometryisavaluabletoolinthefieldofproteomics.Itcanbeusedtoidentifyproteinsthroughvariationsof
massspectrometrytechniques.Themostcommonfirstapproachtoproteomicsisabottomupapproachinwhichtheproteinisdigested
byaprotease,suchatrypsin,andthepeptidesarethenanalyzedbypeptidemassfingerprinting,collisioninduceddissociation,tandem
MS,andelectroncapturedissociation.Oncethepeptidesmasseshavebeendeterminedthemasslistcanbesenttoadatabase,suchas
MASCOT,wherethelistiscomparedtothemassesofallknownpeptides.Ifenoughpeptidesmatchthatofaknownproteinyoucan
identifyyourprotein.Ifthemassesofyourpeptidesdonotmatchaknownproteinyoucansequenceyourpeptidebydenovosequencing
usingMS/MSmethodswhereyouisolateyourpeptideandbreakitalongthepeptidebondbackboneformingyandbionsfromwhich
youcandeterminethesequence.Theadvantagesofthebottomupapproacharethatthesmallsizeoftrypticpeptideionsiseasyto
handlebiochemicallythanentireproteinionsbecauseoftheirrelativelysmallmassesthatareeasiertobedetermine.Besidebottomup
approach,anotherapproachistopdown.Intopdownapproach,thecompleteproteinsaredirectlyanalyzedbyusingmassspectrometer
withoutsolutiondigestionasbottomupdoes.Theadvantagesofthetopdownapproacharethatitcansometimeprovidethecomplete
coverageoftheprotein.Butsincewholeproteinsarehardtohandlebiochemicallycomparedtosmallpeptidepieces,itmakestopdown
approachdifficulttoanalyze.
Anotheruseofmassspectrometryinproteomicsisproteinquantification.Bylabelingproteinswithstableheavierisotopesyoucanin
turndeterminetherelativeabundanceofproteins.Companiesnowproducekits,suchasiTRAQ(AppliedBiosystems)(https://products.a
ppliedbiosystems.com/ab/en/US/adirect/abjsessionid=h5NJJtQYRTsxJ87QQ1gvLQR10ytg61ntRqKBGJdvy2KhJ6zJJD5z!132698344
8?cmd=catNavigate2&catID=600900&tab=Literature),inordertodothisatahighthroughputlevel.
Oneofthemostpowerfulwaystoidentifyabiologicalmoleculeistodetermineitsmolecularmasstogetherwiththemassesofits
componentbuildingblocksafterfragmentation.Therearetwodominantmethodsfordoingthis.Thefirstiselectrosprayionization(ESI),
inwhichtheionsofinterestareformedfromsolutionbyapplyingahighelectricfield.Thisisdonebyapplyingahighelectricfieldto
thetipofacapillary,fromwhichthesolutionwillpassthrough.Thesamplewillbesprayedintotheelectricfieldalongwithaflowof
nitrogentopromotedesolvation.Dropletswillformandwillevaporateinavacuumedarea.Thiscausesanincreaseinchargeonthe
dropletsandtheionsarenowsaidtobemultiplycharged.Thesemultiplychargedionscannowentertheanalyzer.ESIisamethodof
choicebecauseofthefollowingproperties:(1)The"softness"ofthephaseconversionprocessallowsveryfragilemoleculestobeionized
intactandeveninsomenoncovalentinteractionstobepreservedforMSanalysis.(2)Theelutingfractionsthroughliquid
chromatographycanthenbesprayedintothemassspectrometer,allowingforthefurtheranalysisofmixtures.(3)Theproductionof
multiplychargedionsallowforthemeasurementofhighmassbiopolymers.Multiplechargesonthemoleculewillreduceitsmassto
chargeratiowhencomparedtoasinglechargedmolecule.Multiplechargesonamoleculealsoallowsforimprovedfragmentationwhich
inturnallowsforabetterdeterminationofstructure.Thesecondismatrixassistedlaserdesorption/ionization(MALDI)inwhichthe
molecularionsofinterestareformedbypulsesoflaserlightimpactingonthesampleisolatedwithinanexcessofmatrixmolecules.This
enablesthedeterminationofmassesoflargebiomoleculesandsyntheticpolymersgreaterthan200,000Daltonswithoutdegradationof
themoleculeofinterest.TheadvantagesofMALDIareitsrobustness,highspeed,andrelativeimmunitytocontaminantsand
biochemicalbuffers.
AtypeofmassspectrometeroftenusedwithMALDisTOForTimeofFlightmassspectrometry.Thisenablesfastandaccuratemolar
massdeterminationalongwithsequencingrepeatedunitsandrecognizingpolymeradditivesandimpurities.Thistechniqueisbasedon
anultravioletabsorbingmatrixwherethematrixandpolymeraremixedtogetheralongwithexcessmatrixandasolventtoprevent
aggregationofthepolymer.Thismixtureisthenplacedonthetipofaprobethenthesolventisremovedwhileundervacuumconditions.
Thiscreatescocrystallizedpolymermoleculesthataredispersedhomogeneouslywithinthematrix.Apulsinglaserbeamissettoan
appropriatefrequencyandenergyisshottothematrix,whichbecomespartiallyvaporized.Inturnthehomogeneouslydispersedpolymer
withinthematrixiscarriedintothevaporphaseandbecomescharged.Toobtainasuperbsignaltonoiseratio,multiplelasershotsare
executed.Theshapesofthepeaksareimprovedandthemolarmassesdeterminedaremoreaccurate.Fianlly,intheTOFanalyzerthe
moleculesfromasampleareimpartedidenticaltranslationalkineticenergiesbecauseoftheelectricalpotentialenergydifference.These
ionicmoleculestraveldownanevacuatedtubewithnoelectricalfieldandofthesamedistance.Thesmallestionsarrivefirstatthe
detector,whichproducesasignalforeachion.ThecumulativedatafrommultiplelasershotsyieldaTOFmassspectrum,which
translatesthedetectorsignalintoafunctionoftime,whichinturncanbeusedtocalculatethemassoftheion.
Inadditiontotheseionizationtechniques,highlypowerfulmassanalyzershavebeendeveloped.Theseanalyzersmeasurethe
mass/chargeratioofintactionizedbiomolecules,aswellastheirfragmentationspectra,withhighaccuracyandhighspeed.The
measurementoffragmentationspectraiscalledtandemMSorMS/MS.InconjunctionwithsinglestageMS(withintactprecursorions)
tandemMScanbeutilizedtohelpelucidateaproteinsincetheproblemofelucidationwillreducetoassemblingthepuzzlepiecesofthe
fragmentedprotein.
References
AmericanSocietyforMassSpectrometryWhatisMS?,http://www.asms.org/whatisms/p4.html
MassSpectrometryinthePostgenomicEraAnnualReviewofBiochemistryVol.80:239246(VolumepublicationdateJuly2011)DOI:
10.1146/annurevbiochem110810095744https://ted.ucsd.edu/webapps/portal/frameset.jsp?
tab_tab_group_id=_2_1&url=%2Fwebapps%2Fblackboard%2Fexecute%2Flauncher%3Ftype%3DCourse%26id%3D_767_1%26url%3D
UniversityofIllinoisatUrbanaChampaignSchoolofChemicalScienceshttp://scs.illinois.edu/massSpec/ion/esi.php
UniversityofSouthernMississippiSchoolofPolymersandHighPerformanceMaterialshttp://www.psrc.usm.edu/mauritz/maldi.html
ProteinPrimaryStructure
Presentation
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Introduction
PosttranslationalModification
Presentation
Printversion
Introduction
I.Definition
A.Spontaneousorenzymaticalterationtooneormoreofaprotein'saminoacids
B.Mostoftenmanifestsasanadditionordeletiontoasidechain
C.Canoccuratanypointduringorfollowingfulltranslationofaprotein
D.Oftendrasticallyeffectsoverallstructureandfunctionofproteinandassociatedcomplexes
E.Arehighlyconservedamongalllivingorganisms
II.Typesofmodifications
A.Acetylation
B.Amidation/Deamidation
C.Glycosylation
D.Oxidation
i.SGlutathionylation
ii.SNitrosylation
E.Phosphorylation
i.Histidine
ii.Serine
iii.Theronine
iv.Tyrosine
F.Proteolysis
G.Ubiquitinylation/SUMOylation
H.Others?
III.Manipulatinginvivomodifications
A.Modificationscanbepreventedorinducedinorganism,tissue,andcellbasedmodelsystems
B.Mayallowforthedetectionoftargetproteinsordissectionofrelatedprocessesandpathways
C.Exogenousintroductionofstimuli
i.endocrine/paracrinesignals(akahormones)
ii.environmental(temp,UV,heavymetals,peroxide,etc.)
iii.antigens(virus,bacteria,allergens,lysates,etc.)
iv.chemical/medicinalactivatorsandinhibitors
D.GeneticApproaches
i.deletion,mutation,orreorganizationofgeneticelements(enhancers,promoters,genes,etc.)
ii.geneinactivationorsilencingbynucleicacidhibridization
iii.transgenics(tansformationsandtransfections)
E.AdvantagesandDrawbacks
i.Qualitativebutoftenhardtoquantify
ii.Oftenexpolaratoryinnature(observeandreport)
iii.Oftenverylargescaleintermsofavailabledata
IV.Invitroreconstitutionstrategies
A.Oftenprovideamorequantitative,indepthanalysisofaparticularposttranslationalmodification
B.Theproteininquestionisaddedtoareactionwiththeappropriatereagentsand/orenzymes
C.Reactionscanbefollowedinrealtimemorereadily
V.Methodsofdetection
A.Mostcommonlyuseddetectionmethodofknownmodificationsisthroughimmuno/Westernblotting
B.Unexpectedornovelmodificationscanbedetectedwithavarietyofanalyticaltechniques,mostnotablymassspectrometry
C.Thebestspecificmethodofdetectiondependsonmanyfactorsincludingthestability,frequency,andscaleofthemodification(s)
D.Inpracticeeaseandcostdictatewhichmethodsareusedfirstwhilethemoreexhaustive,cumbersome,orexpensivemethodsfollowasneeded
ProteinProteinInteractions
Presentation
Printversion
Chaptereditedandupdatedby:PoulamiBarmanandSarahAllen
Contactpxb2979@rit.edu,sea3016@rit.edu
Introduction
Proteininteractioniscrucialforeveryorganism.Mostproteinsfunctionthroughinteractionwithothermolecules,andoftenwithother
proteins.Enzymesinteractwiththeirsubstrates,inhibitorsinteractwithenzymes,transportproteinsinteractwithstructuralproteins,
hormonesinteractwithreceptorsandthatsjustafewoftheinteractionsthathappeninacell.Someproteinsarecomposedofmore
thanonepolypeptidechain,andtheinteractionsbetweenthedifferentpeptidesarenecessaryforthewholeproteintofunction.Sincethey
aresoessential,proteinproteininteractionsareanimportanttopicforscientiststounderstand.
Therearemanycharacteristicsofaproteinproteininteractionthatareimportant.Obviously,itisimportanttoknowwhichproteinsare
interacting.Inmanyexperimentsandcomputationalstudies,thefocusisoninteractionsbetweentwodifferentproteins.However,you
canhaveoneproteininteractingwithothercopiesofitself(oligomerization),orthreeormoredifferentproteinsinteracting.The
stoichiometry(http://en.wikipedia.org/wiki/Stoichiometry)oftheinteractionisalsoimportantthatis,howmanyofeachprotein
involvedarepresentinagivenreaction.Someproteininteractionsarestrongerthanothers,becausetheybindtogethermoretightly.The
strengthofbindingisknownasaffinity.Proteinswillonlybindeachotherspontaneouslyifitisenergeticallyfavorable.Energychanges
duringbindingareanotherimportantaspectofproteininteractions.Manyofthecomputationaltoolsthatpredictinteractionsarebasedon
theenergyofinteractions.
Inrecentyearstherehasbeenastrongfocusonpredictingproteininteractionscomputationally.Predictingtheinteractionscanhelp
scientistspredictpathwaysinthecell,potentialdrugsandantibiotics,andproteinfunctions.However,ithasbeenanongoingchallengeto
decipherthoseinteractions.Proteinsarelargemolecules,andbindingbetweenthemofteninvolvesmanyatomsandavarietyof
interactiontypes,includinghydrogenbonds(http://en.wikipedia.org/wiki/Hydrogen_bonds),hydrophobicinteractions(http://en.wikipedi
a.org/wiki/Hydrophobic_interactions),saltbridges(http://en.wikipedia.org/wiki/Salt_bridge_%28protein%29),andmore.Proteinsare
alsodynamic,withmanyoftheirbondsabletostretchandrotateleadingtodifferentconformations.Therefore,predictingproteinprotein
interactionsrequiresagoodknowledgeofthechemistryandphysicsinvolvedintheinteractions.
Thischapterdiscussesthecharacteristicsofproteinproteininteractions,howtheyaredeterminedexperimentally,andhowtheyare
predictedcomputationally.Italsocontainsalistofdatabaseswhereyoucanexploreknownandpredictedproteininteractions.Thelinks
abovewillleadyoutothevarioussections.
ProteinChips
Presentation
Printversion
Introduction:
Introduction
Proteinchips,alsoreferredtoasproteinarraysorproteinmicroarrays,aremodeledafterDNA
microarrays.ThesuccessofDNAmicroarraysinlargescalegenomicexperimentsinspired
researcherstodevelopsimilartechnologytoenablelargescale,highthroughputproteomic
experiments.Proteinchipsenableresearcherstoquicklyandeasilysurveytheentireproteomeof
acellwithinanorganism.Theyalsoallowresearcherstoautomateandparallelizeprotein
experiments.
Proteinchipswerefirstdevelopedin2000byresearchersatHarvardUniversity.[1]Todaythere
aremanycompaniesmanufacturingproteinchipsusingmanytypesoftechniquesincluding
spottingandgelmethods.Thetypesofproteinchipsavailableinclude"labonachip",antibody
arraysandantigenarrays,aswellasawiderangeofchipscontaining"alternativecaptureagents"
suchasproteins,substratesandnucleicacids.
Analysisofproteinchipscomeswithmanychallengesincludingdynamicproteinconcentrations, ADNAmicroarrayasseenthrougha
thesheernumberofproteinsinacell'sproteome,andtheunderstandingoftheprobesforeach microscope.Proteinchipslook
protein.Stepsincludethereadingoftheproteinlevelsoffthechip,andthentheuseofcomputer identical,excepteachspot
softwaretoanalyzethemassiveamountsofdatacollected. correspondstooneoftheorganism's
thousandsofproteins,insteadofone
Applicationsofproteinchipexperimentsincludeidentifyingbiomarkersfordiseases, ofit'sgenes.Theintensityofthedot
investigatingproteinproteininteractions,andtestingforthepresenceofantibodiesinasample. indicatestheamountofprotein
Proteinchipshaveapplicationsincancerresearch,medicaldiagnostics,homelandsecurityand present.
proteomics.
Thischapterwilldemonstratewhyproteinchipsarechangingthefaceofproteomics,andwhytheywillhaveanevenlargerimpactinthe
future.
History
NucleicAcidMicroarrays
Theuseofmicroarraysforgeneexpressionprofilingwasfirstpublishedin1995.[2]Thistechnologyallowedscientiststoanalyze
thousandsofmRNAsinasingleexperimenttodeterminewhetherexpressionisdifferentindiseasestates.Unfortunately,mRNAlevels
withinacellareoftenpoorlycorrelatedwithactualproteinabundance.[3]Thiscanbeduetomanyfactorsincludingdegradationrateof
mRNAversusproteinsandposttranscriptionalcontrolsandmodifications.Measuringtheamountofproteindirectlywouldbypassany
mRNAinconsistenciesandgiveatruelevelofgenefunction,howevertraditionalproteincharacterizationmethodswereslowand
cumbersome.Thesecombinedfactorsweretheimpetusbehindthecreationofproteinchips.
DeficiencyofTraditionalProteinCharacterizationMethods
Beforetheadventofproteinchips,proteinmeasuringandcharacterizationwasdoneusingtwo
differentmethods:2Dgelelectrophoresiscoupledwithmassspectrometry,andliquid
chromatography.Thesemethodscanseparateandvisualizealargenumberofproteinsper
experiment,howevertheyaretimeconsumingwhencomparedtoproteinchips.Theirprocessis
verylowthroughputbecauseoflackofautomation.Reproducibilityisalsoafactorbecauseofthe
largeamountofsamplehandling.Abetter,morestandardized,higherthroughputmethodneeded
tobeinventedforproteinmeasuringandcharacterization.
ProteinChipPrecursorstoModernDay
Aliquidchromatography/mass
Immunoassays,theprecursortoproteinchipsavailablesincethe1980s,exploittheinteractions spectrometry(LC/MS)instrument.
betweenantibodiesandantigensinordertodetecttheirconcentrationsinbiologicalsamples. Thistechniqueislowthroughput
Theircreation,however,istediousandexpensive.Asaresponsetothis,researchersatHarvard comparedtoproteinchipsbecause
UniversitycombinedthetechnologiesofimmunoassaysandDNAmicroarraystodevelopthe proteinchipscantestforthousandsof
proteinsonasinglechipinasingle
proteinchip.[4]Intheirlandmarkpaper,publishedin2000,"PrintingProteinsasMicroarraysfor experiment.
HighThroughputFunctionDetermination,"GavinMacBeathandStuartSchreiberdescribedhow
tocreateproteinchipsanddemonstratedthreetypesofapplicationsthatwouldbenefitfromthis
newtechnology.Thestrengthsoftheirapproachweretheuseofreadilyavailablematerials(i.e.
glassslides,polyacrylamidegel),therelativeeaseofimplementation(roboticmicroarray
printers),andcompatibilitywithstandardinstrumentation.
Withinthepastfiveyears,manycompanies,includingBiacore(http://www.biacore.com),
Invitrogen(http://www.invitrogen.com),andSigmaAldrich(http://www.sigmaaldrich.com),have
begunproductionofindustriallevelproteinarraysystemsthatcanbeusedfordrugdiscoveryand
basicbiologicalresearch.Commercialentitieshavemadeproteinchipresearchastreamlinedand
standardizedprocessonthesamelevelasDNAmicroarrayscomparedtoitsinceptionin2000. Theequipmentandreagentsusedin
anEnzymelinkedImmunosorbent
Academicresearchplaysahugeroleinthedevelopmentandimprovementofthesetechnologies. Assay(ELISA),aprecursorof
ThecollaborationofacademicresearchwithsystemssuchastheAffymetrixGeneChipandthe proteinchips.
HumanGenomeInitiativehasallowedforfriendlycompetition,resultingintheadvancementof
technologies.Withmoredevelopscomeabetterunderstandingandencouragesevenmore
researchtowardsthesefields.
Affymetrix(http://www.Affymetrix.com)isacompanythathasbeenmanufacturesmicroarrays,namedGeneChip,since1992.Theyhave
13locationsacrosstheworldwithheadquarterslocatedintheUS(California),UK,Japan,andChina.[5]
Nextsection:Manufacture
References
1.MacBeathG,SchreiberS.(2000).PrintingProteinsasMicroarraysforHighThroughputFunctionDetermination.Science.Sep08
289(5485):17601764.
2.SchenaM,ShalonD,DavisRW,BrownPO.(1995).Quantitativemonitoringofgeneexpressionpatternswithacomplementary
DNAmicroarray.Science.Oct20270(5235):46770.
3.GygiSP,RochonY,FranzaB,AbersoldR:CorrelationbetweenproteinandmRNAabundanceinyeast.Mol.CellBiol.19,1720
1730(1999).
4.MacBeathG,SchreiberS.(2000).PrintingProteinsasMicroarraysforHighThroughputFunctionDetermination.Science.Sep08
289(5485):17601764.
5."Affymetrix."Wikipedia,TheFreeEncyclopedia.5Feb2007,03:19UTC.WikimediaFoundation,Inc.Apr2008
<http://en.wikipedia.org/wiki/Affymetrix>
Chapterwrittenby:JonathanKeelingandEricFoster
Contactjwk3970@rit.edu,edf3480@rit.edu,ttl5439@rit.edu
ProteomicsandDrugDiscovery
Presentation
Printversion
Chapterwrittenby:PiotrKowalskiandPatrickKenney
Contact:pxk9006@rit.edu,pok7810@rit.edu
ThisSection:
IntroductiontotheDrugDiscoveryProcess
Theprocessofdrugdiscoverywithinthemodernscientificcontextisquitecomplex,integratingmanydisciplines,includingstructural
biology,metabolomics,proteomics,andcomputerscience,justtonameafew.Theprocessisgenerallyquitetediousandexpensive,
giventhesheeramountofpossibilitiesofdrugtotargetinteractionsinvivo,andthenecessityofsuccessfullypassingrigorous
pharmacokineticstudiesandtoxicologyassayspriortoevenbeingconsideredforclinicaltrials(Burbaum).Thoughamoredetailed
explanationisofferedfurtherintothistext,severalkeycomponentsofthedrugdiscoveryprocessincludetargetselection,lead
identification,andpreclinicalandclinicalcandidateselection.Theschematicontherightoutlinesthestepsinvolvedinthedrug
discoveryprocess.
Therecentboomoftheproteomicsfield,ortheanalysisoftheeverdynamicorganismalproteome,hasbroughtmanyadvanceswith
respecttotheverynatureofhowthecurrentdrugdiscoveryprocessisundertaken.Thepotentialthefieldofproteomicsbringsinfor
identifyingproteinsinvolvedindiseasepathogenesisandphysiologicalpathwayreconstructionfacilitatestheeverincreasingdiscovery
ofnew,noveldrugtargets,theirrespectivemodesofactionmechanistically,andtheirbiologicaltoxicology(Page).
Thechallengeinthedrugdiscoveryprocessistofindtheexactcausesofanunderlyingdiseaseandfindawaytonegatethemorbring
themtonormallevels.Amechanisticunderstandingofthenatureofthediseaseinquestionisessentialifwearetoelucidateanytarget
specificremedyforit.Whilethecausesofmanydocumentedclinicalproblemsvarygreatlyintheirnatureandorigin,insomecases,the
causeisfoundattheproteinlevel,involvingproteinfunction,proteinregulation,orproteinproteininteractions.Oneexampleofsucha
disorderwouldbealkaptonuria,characterizedbyadefectinthegenecodingfortheenzymehomogentisicacidoxidase[16](http://www.e
xpasy.org/cgibin/nicezyme.pl?1.13.11.5),inhibitingthemetabolismofhomogentisicacidtomaleylacetoaceticacid,withinthe
phenylalaninedegradationpathway(Brooker).Whiletheunderlyingcauseofthisinborndiseaseisduetoasinglegenegeneticdefect,
theclinicalmanifestations,whichincludeexcretionofblackurine,areafunctionofthebuiltupofhomogentisicacidresultingfroma
defective[protein]enzyme.
Recentadvancesinappliedgenomicshelpedinthetargetidentificationprocess,sinceitallowedforhighthroughputscreeningof
expressedgenes.However,studieshaveshownthatthereisapoorcorrelationbetweentheregulationoftranscriptsandactualprotein
quantities.Thereasonsforthisarethatgenomeanalysisdoesnotaccountforposttranslationalprocessessuchasproteinmodifications
andproteindegradation.Therefore,themethodsemployedinthedrugdiscoveryprocessstartedtoshiftfromgenomicstoproteomics
(Burbaum).Analysisofthedynamicorganismalproteome,asopposedtothestaticgenome,willcertainlybringamuchmoreaccurate
approachtoidentifyingnotonlyapplicablebiomarkersthatwillaidindiagnosis,butalsoeffectiveremediesfordiseasesofvarying
origins.
Thefieldofproteomicsfacessomedauntingchallenges,incomparisontogenomics,forseveralreasons.First,proteinsciencelacksan
analogueofthepolymerasechainreaction(PCR),whichcangeneratemanycopiesofasingle,nativemoleculeinvivo(nucleicacidsin
thecaseofPCR).However,severalrecentapproacheshavebeenappliedinanefforttoamelioratethisquandary.Methodsofchemical
synthesisexist,beinglimitedbyyield,particularlywhenitcomestosynthesizinglengthypeptides.Invivoexpressionsynthesismethods
existaswell,however,thisapproachcannotbeappliedtoproducingproteinswhichmayalternormalcellularfunction.Also,cellfree
synthesisribosomekitscanalsobeemployedforaccurateandrapidproteinsynthesis,thoughtheintrinsicpresenceofribosome
inactivatingenzymescontributestotheinstabilityofthesesystems(Madin).Second,incontrasttoDNA,proteinlevelsvarysignificantly
dependingoncelltypeandenvironment.Third,proteinabundanceisnotdirectlycorrelatedtoproteinactivity.Proteinactivityisoften
determinedbyposttranscriptionalmodificationssuchasphosphorylation.Proteinactivity,notproteinabundance,isofinterestinthe
drugdiscoveryprocess.Finally,proteinsformmanyinteractionswithother
proteinsorsmallmolecules.Elucidationoftheseinteractionswouldgreatly
speedupthedrugdiscoveryprocess.Onewaythisiscurrentlybeingdoneis
throughligandboundxraycrystallographicstudies.
Theidealproteomicstechniquesuitedfordrugdiscoverywouldhavethe
followingfeatures:itshouldbeabletoseparatemembraneproteinsanddetect
lowabundanceproteins,twoabilitiesnotquiteyetrealized,yetrequiredin
currentseparationsandanalyticaltechniques.Furthermore,itshouldbeableto
identifyproteinactivityindependentofproteinabundance.Italsoshouldreveal
proteinproteinandproteinsmallmoleculeinteractions.Thismethodshouldalso
beimplementedeasily,beautomatable,andperformathighthroughputspeed.
Proteomicsresearchersareaddressingtheseissues,andnewmethodsarebeing
developed(Burbaum).
Virtualdruglibrariesarebeingdeveloped,bothinthepublicandprivatesectors.
Thesedatabasescontainpotentialdrugcompoundsthesecompoundsmayor
maynotexistoutsideofacomputerdatabase,andnewcompoundsdeveloped
throughvariousmethodsofsynthesisarecontinuallyadded.Methodsof
modifyingexistingdatabaseentriestocreatenewisomersandderivativesare
alsoused,tomoreadequatelycoverarangeofpotentialdrugcompounds.
Dockingandscoringareimplementedusingknownandhypotheticaldrugtargets
onaprotein,coupledwiththedatabasesofvirtualchemicalcompounds.In
docking,variouscomputationalmethodsareusedtopositionachemicalproperly
withinaproteinbindingsite.GeneticalgorithmsandMonteCarlomethodsare
twopopularalgorithmsforevolvinganoptimumbindingposition.Thisprocess
screensforchemicalsthatarepotentialdrugs,whichinitiallyaretermedashits.
Afterdocking,scoringiscarriedoutusingmathematicalmodels.Thesemodels
determinethechemicalbindingstrengthandenergystateofthedrugprotein
complex.Thosehitswithhighrankingscoresaresuqsequentlysubjectedtoin
vivotestshitswithpositivescoresinbothareasarethenknowntobeleads
(Bleicher).
Evaluationofdockedandscoredcomplexesarethenmade,selectinganarbitrary
numberoftophitstobefurtherscreenedmanually.Thefirsttwostepsaredone
entirelyinsilicohowever,thebestcomplexesnowneedtobeexaminedusing
softwarevisualization,ofteninthreedimensionalsetups.Thisallowsscientists
toensurethatthedetermineddockingorientationlooksacceptable,andthatthe
scoringiscorrectbasedonknowninteractionenergiessuchashydrogenbondsandionicinteractions.
Thecompoundsthatmakeitthroughdocking,scoring,andevaluationbecomedrugleads,andarethenpassedontoundergodrugtesting
techniquesbyscientistsinawetlab,toensurethatonlycompoundswitheffectsrelativelyuniquetothetargetsystemandsafetotherest
oforganismareconsidered.However,thedrugcompanyhasalreadysavedmuchtimeandmoneyuptothispointbyhavingcomputers
dochemicalscreening,ratherthanhumanscientists.
References:
OpenAccessArticles
Burbaum,Jonathan,etal.HighResolutionFunctionalProteomicsbyActivesitePeptideProfiling.PNAS.2005Mar:(102),49965001.
[[17](http://www.pnas.org/cgi/reprint/102/14/4996.pdf)]
Madin,Kairat,etal.Ahighlyefficientandrobustcellfreeproteinsynthesissystempreparedfromwheatembryos:Plantsapparently
containasuicidesystemdirectedatribosomes.AppliedBiologicalSciences.2000Jan97(2),559564.
SubscriptionRequiredArticles
Bleicher,H.Konrad,etal.HitandLeadGeneration:BeyondHighThroughputScreening.NatureReviews,DrugDiscovery.2003May:
(2),369378.
Brooker,J.Robert.Genetics:AnalysisandPrinciples.Benjamin/CummingsPublishers:MenloPark,CA,1999.1sted.,pp.316317.
Page,J.Martin,etal.Proteomicsasamajornewtechnologyforthedrugdiscoveryprocess.DrugDiscoveryToday.1999Feb:(4),2,55
62.
Next:RationalDrugDesign
Biomarkers
Presentation
Printversion
Massspectrometrybasedtargetedproteinquantification:methodsandapplications(http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC2657955/?tool=pmcentrez)
MainFocus File:TripleQ.jpg
Crosssectionoftriple
Themainfocusofthepaperwasareviewofthemethodsandapplicationsofusingmassspectrometryto
Q
quantifyproteins,especiallythosethatareinaconcentrationoflessthang/mlconcentrations,inanattemptto
universalizeaprocedure.
Newterms
MALDITOF/TOF
matrixassistedlaserdesorption/ionizationtimeofflighttandemmassspectrometer.
Selectedreactionmonitoring(SRM)
methodinwhichaspecificproductionfromaspecificparentionisdetected.Allotherionswithmassesnotspecifiedina
predeterminedrangearefilteredawayleavingonlyionswiththemassintherangewearelookingfor.(source
http://en.wikipedia.org/wiki/Mass_chromatogram#Selected_reaction_monitoring_.28SRM.29)
Triplequadrupole
typeofMSthatcontainsalinearseriesofthreequadrupoles.Thefirstandthirdsetactasmassfilters,andthesecondisacollision
cell.ThistypeofMScanfilteranionofaknownmass.(sourcehttp://en.wikipedia.org/wiki/Quadrupole_mass_analyzer)
Hydrazide
classoforganiccompoundsthatshareacommonfunctionalgroupcharacterizedbyaNNcovalentbondwithoneofthe
constituentsbeinganacylgroup.(sourcehttp://en.wikipedia.org/wiki/Hydrazide)
Biomarker
biochemicalfeaturethatcanbeusedtomeasureprogressoftheeffectsoftreatmentoradisease.(source
http://www.medterms.com/script/main/art.asp?articlekey=6685)
Forthissummary,wewillfocusonproteinbiomarkers.Somediseaseswhichhaveproteinbiomarkersthatshowpromiseasascreening
toolarebreastcancer,Alzheimer's,leukemia,ALS,andParkinson's[1].Aseriesofsixstepsmustbeaccomplishedinorderto
successfullyvalidateabiomarkerorsetofbiomakers:discovery,qualification,verification,assayoptimization,validationand
commercialization[2].Onceabiomarkerisfoundandaccepted,itcanbeusedtopossiblypredictandpreventthediseaseit'srelatedto.
Thesummarybelowfocusesonthequantificationmethodofproteinsinthesearchforandidentificationofproteinbiomarkers.By
findingwaysinwhichtouniversallyquantifyproteins,onecansearchforallbiomarkersinonescreeningratherthanmultiplescreenings,
onceconclusivebiomarkersareidentified.
Summary
Withtherecentbreakthroughsintechnology,itisconceivablethatispossibletohaveauniversalmethodorapproachwithminimal
restrictionstoquantitativelyassayawidenumberofproteinsinsearchofpotentialbiomarkers.Onceafewpotentialbiomarkersare
discovered,furtherresearchcanbedonetoconfirmorrefuteitsuseinclinicalapplications.Anothergoalistoeasilyaccumulatemultiple
detectionsinasinglemeasurement.Measurementsaretakenbyidentifyingsyntheticallystableisotopesattachedtotheirrespective
proteinsorpeptides.Eachisotopemimicsthepeptidesendogenouscounterpartsallowinghighselectivity.
Massspectrometry(MS)providesuswithapowerfultooltocomparetwodifferentproteinsamples.Itcanbeusedforcomparingthe
proteomeofadiseasedsampleagainstanormalsampleataglobalscale.Thisisappliedtoawidearrayofhumandiseases,withthehope
thatitwillleadtoidentificationofbiomarkersorevenpathogenesisofadisease.Traditionally,ELISA(http://en.wikipedia.org/wiki/ELIS
A)(enzymelinkedimmunosorbentassay)hasbeenthemajormethodforthequantificationofproteinswithgoodsensitivity.Eventoday,
itisthegoldstandardfortargetedproteinquantification.ThemajordrawbackwithELISAisthelackofavailabilityofantibodieswith
highspecificity.
FirstattemptstodeterminetheamountofspecificproteinsweredoneusingstableisotopedilutionmethodsandMSapproximately20
yearsago,startingwithatombombardmentMSanddeuteriumlabeledsyntheticpolypeptides.AdvancesinMSinstrumentationhas
increasedourabilitytodetectcandidateproteinsincomplicatedbiologicalsampleswithhighsensitivity.Toquantifytheresults,
introductionofastableisotope(containing13Cor15N,forexample)toselectedaminoacidsinareferencepeptidesequenceprovidesa
peptidewiththesamephysicochemicalproperties,thatcanbereadilydistinguishedbyMSfromthepeptideinthetargettissueorfluid.
Studieshaveshownthatfulllengthproteinswithstableisotopescanbeusedinquantificationofbiomarkersinurineandwatersamples
withnanomolarandpicomolarlevelsensitivitiesrespectively.
ThereisavarietyofMSplatformsusedforquantitativeproteomics,someofwhicharetriplequadrupole(http://en.wikipedia.org/wiki/Qu
adrupole_mass_analyzer#Triple_quadrupoles)(tripleQ),matrixassistedlaserdesorption/ionizationtimeofflighttandemmass
spectrometer(MALDITOF/TOF(http://en.wikipedia.org/wiki/MALDITOF#Mass_spectrometer)),electrosprayionization(ESI)based
onQTOFMS,andaniontrapinstrumentusingselectiveionmonitoring(SIM)mode.Themostpopularoftheplatformsaboveisthe
tripleQ.Demonstrationshaveshownthatitcanmultiplexandsimultaneouslytargetmorethan50peptidesforquantificationinplasmain
asinglemeasurement.Fortargetedquantitativeanalysis,couplingliquidchromatographywithMALDIgreatlyenhancestheperformance
ofMALDIbasesMS.Someadvantagesofthisapplicationincludetheabilityofthetechniquestobeperformedinparallelwitheach
other,itcanbemadeahighlyselective,datadrivenprocedure,andthepreservationofthesampletosomedegreeforrepeatanalysis.
Thistechniqueisalsohighlightedbyitspotentialhighthroughputandexcellentresolution.
Oneofthemostimportantstepsinquantificationissamplepreparationwhichgreatlyinfluencessensitivity.Oneofthemostcommon
stepsusedisthedepletionofhighlyabundantproteinsmakingiteasiertoenhanceanalyticaldynamicrangeandthedetectionofproteins
inlowconcentrations.Oneofthetechniquesperformedisstrongcationexchangechromatography(SCX)whichhasshowntogivethe
abilitytodetectpeptidesinthehighpg/mllevel,givinga100foldimprovementoverdirectplasmaanalysis.
Posttranslationalmodification(PTM)isanimportantprocesstounderstandsinceitisofteninvolvedintumorprogression,butcanbea
problemtomimicduetothecomplexityandstructureofthesugarchains(asinglycosylationPTM).OneexperimentextractedNlinked
glycopeptidesanddeglycosylated.ThisresultedintheconversionofAsntoAspandadifferenceinmass.Thiswasutilizedtomakea
syntheticpolypeptidetoreplicateanNlinkedglycopeptideinitsglycosylatedform.
OnceofthemainfeaturesofMSbasedquantificationisforclinicalapplicationsusedtoidentifybiomarkersassociatedwithdiseases.For
example,177proteincandidatesassociatedwithstrokeandcardiovasculardiseaseinplasmahavebeenproposed.Somebiomarkers
affiliatedwithstrokeareS100b,Btypeneurotrophicgrowthfactor,vonWillebrandfactorandmonocytechemotacticprotein1[3].Other
biomarkershavebeenproposedtorheumatoidarthritisandbreastcanceramongothers.
Oneofthemaingoalsoftheabilitytoquantifyproteinsandpeptidesisforpersonalizedmedicine.Astechnologyadvances,wewillbe
abletocreatetechniquesthateasilyassemblemultiplesdetectioninasinglemeasurement.Biomarkersfromdiseasescanalsobe
multiplexedinasingleassay,allowingustopossiblydiagnosemultiplediseases.Ideally,asinglesteppreparationstrategyiskey,
allowingforhighthroughputandpossibleanautomatedprocess,reducingtheamountofhumaninteractionandthechanceofhuman
error.
RelevancetoProteomicsCourse
Theabilitytoquantifyproteinsusingmassspectrometryisagreattooltocomparealargenumberofproteinsfromacontrolsamplewith
atestsampleinsearchofbiomarkers.Whenanoticeabledifferenceisdetected,furtherstudiescanbeperformedonthoseproteins.Major
breakthroughsinMStechnologygiveusthecapabilitytouniversallyapproachdevelopmentstoquantifyawidespectrumofproteins
withlittlerestriction.Italsogivesustheabilitytomakemoredetectionspermeasurement.Inthefuture,theseapproachescangiveway
topersonalmedicinegivingustheabilitytoscreenindividualsbydetectingmultiplebiomarkersfromasingleormultiplediseases.
References
[1]PharmaceuticalOutsourcingDecisions.SPGMediaLimited.
(http://www.pharmaceuticaloutsourcing.com/articles/pod003_014_power3.htm)
[2]Rifai,Nader,Gillette,MichaelA.,andCarr,StevenA."Proteinbiomarkerdiscoveryandvalidation:thelonganduncertainpathto
clinicalutility".NatureBiotechnology24,971983(2006)(http://www.nature.com/nbt/journal/v24/n8/abs/nbt1235.html)
[3]Reynolds,MarkA.,etal."EarlyBiomarkersofStroke".ClinicalChemistry49(2003):17331739.Print.
(http://www.clinchem.org/cgi/content/abstract/49/10/1733)
ExperimentalProtocols
Presentation

Printversion
Thispagecontainsprotocolsthatarefrequentlyusedinproteomics.Youarewelcometoaddprotocolsthischapter.
1.PlantProteomicsaboutTwoDimensionalGelElectrophoresis
Contributors
Contributorslist
Pleasefeelfreetoaddyournameandcontactinformationifyouwouldliketoberecognizedforyouradditiontothispageorcontacted
byothersintheproteomicscommunity.
TungLuong(http://tungluong.blogspot.com/%7C)tung.luong gmail.com
AdamCornwellibmman85 gmail.com
AlexanderButarbutarnbb3924 rit.edu
AndresJavierGonzalezajg3600 rit.edu
AnthonyEspositoage5719 rit.edu
AubreyBaileyaubreybailey gmail.com
JaredCarterAdarzaBioSystems,Inc
JohnBoutellJDBoutell gmail.com
JohnBrothersIIjfb4497 rit.edu/jb2 bu.edu
LauraGrellllg3875 rit.edu
LeightonIngleighton.ing gmail.com
LukasHabeggerlh9357 rit.edu
MelissaWilbertmlw3559 rit.edu
MitulPatel(Bioinformatician)mitul428 gmail.com
PatrickKenneypoksch rit.edu
PiotrA.Kowalskipxk9006 rit.edu
TomMaxonTomMaxon gmail.com
VishalThovaraivvt1936 rit.edu
SophiaLafergolaDieselSandwich(talk)sophia.lafergola gmail.com
Template:Pharmaceuticaloutsourcing
Rifai,Naderetal.(2006).Proteinbiomarkerdiscoveryandvalidation:thelonganduncertainpathtoclinicalutility.Nature
Biotechnology.
Reynolds,MarkA.etal.(2003).EarlyBiomarkersofStroke.ClinicalChemistry.
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