ISOLATION OF PROTEIN ISOFORMS BY FREE FLOW ELECTROPHORESIS

PRESERVING BIOLOGICAL ACTIVITY

A. Abdolzade-Bavil,
C. Obermaier, S. Hirler, U. Sukop, M. Nissum, C. Dufter, G. Weber, C. Eckerskorn
BD Diagnostics, Am Klopferspitz 19a, 82152 Planegg, Germany

afsaneh_abdolzade-bavil@europe.bd.com

Title Native-PAGE. L-LDH activity assays confirmed

ISOLATION OF PROTEIN ISOFORMS BY FREE that the biological activity of the sample was
FLOW ELECTROPHORESIS PRESERVING preserved after FFE separation. The binding
BIOLOGICAL ACTIVITY activities of CD3 antibody isoforms were analyzed
using flow cytometry.
Introduction
Protein isoforms are defined as variants of a single Innovative aspects
polypeptide which generally alter its function. More • FFE enables native separation conditions
than 90% of naturally occurring isoforms arise from preserving biological activity of the sample.
post translational modifications (PTMs) and less • The high resolution of FFE, separating
than 10% from mRNA splice variations. proteins based on charge, is ideally suited for
In many cases, also recombinant proteins consist difficult challenges like separation of protein
of several isoforms due to differences like variation isoforms beyond the limitation of Ion-exchange
in the glycosylation pattern or modifications at the Chromatography.
N-terminus. Additionally, several chemical
modifications may occur during protein isolation
and separation processes. Modifications such as
PEGylations may also be introduced chemically
and isoforms are generated through incomplete
reactions.
In many cases PTMs change the biological activity
of proteins. We introduced FFE to isolate individual
protein isoforms under native conditions for further
biological studies. Using Free Flow
Electrophoresis (FFE), sorting out proteins based
on charge, we separated and characterized
multiple isoforms of different samples. Resulting
fractions were well suited both for separation of
closely related species such as protein isoforms,
and for direct use in further studies such as
enzyme assay and/or immunoassays.

Methods
Protein samples were diluted with the appropriate
FFE separation buffers and loaded via sample inlet
to the separation chamber. The FFE separations
were performed under native conditions using a
voltage of 900-1200 V depending on the current
separation.
Continuous Isoelectric Focusing FFE (CIEF)
buffers were prepared according to manufactures
description (BD™ FFE).
CIEF was performed at 10°C with buffer flow rate
between 30-60 ml/hr
Native isoelectric focusing polyacrylamid gel
electrophoresis (IEF-Native-PAGE) was done
using IEF-Gels from Invitrogen and Serva
according to manufactures description.

Results
In this study we separated protein isoforms from
human Anti-CD3, rabbit L-LDH, Amyloglucosidase
from Aspergillus Niger, ß-Lactoglobulin from
bovine milk and bovine plasma Fetuin by FFE.
Obtained FFE fractions were analyzed by IEF-
 A simple way to use a 1D split-free nano-HPLC in an automated 2D LC-MS/MS setup
1 1,3 2 2 2 3
Taylor, P. , Moran, M , Podtelejnikov, A. , Andersen, M. , Vorm, O. and Kislinger, T.
1. Hospital for Sick Children, Ontario, Canada 2. Proxeon, Odense, Denmark 3. University of Toronto,
Department of Medical Biophysics, Ontario, Canada

Introduction
Analysis of complex biological samples often
requires several separation steps. One of the
commonly used technologies in the filed of
proteomics is automated two-dimensional Liquid
Chromatography coupled with Mass Spectrometry.
(2D LCMS) based on on-line strong cation
exchange (SCX) / reverse phase (RP) separations.
That sophisticated system uses ternary or
quaternary gradient generation systems to deliver
the stepped salt concentrations needed to affect
release of the peptides from the SCX resin onto
the RP material and then to elute the peptides
from the RP column followed by MS/MS analysis.
Here we describe a simple, automated SCX/ RP
on-line 2D separation strategy achieved on a split-
free 1D nano LCMS system.

Method
To evaluate the system performance we analyzed
the proteome of mouse placental cells. The
cytosolic fraction was subjected to cysteine
reduction and alkylation with iodoacetamide
followed by trypsin digestion. The lysate was
purified and analyzed by 1D and 2D methods on a
split-free nano LCMS system (EASY n-LC Proxeon
Biosciences, Odense Denmark) coupled to LTQ-
Orbitrap (Thermo-Fisher, Bremen, Germany). In
order to perform 2D separation 10 step injected
salt gradient was used varying from 0.05 to 0.5 M
NH4Acetate.

Results
By using the auto-sampler component and
standard sample injection programs we were able
to accomplish the salt delivery using a split-free
nano LCMS system and therefore transforming the
system into 2D system. The viability of this method
was demonstrated by comparative analysis LCMS
data obtained from 1D and 2D separations. As a
result a 1D analysis yielded about 600 protein
identifications whereas 2D analysis gave
approximately 2000 proteins identified. The
transformation of 1D system into 2D system
makes it possible to obtain much more information
out of a single sample as easily as a traditional 1D
run and making use of the same 1D nano LCMS
setup.

Innovative aspects
These results clearly demonstrate the significant
improvement which can be achieved by slight
modifications of the 1D nano LCMS
 A New Stain Free Gel System and its Compatibility with Western Blotting and Mass
Spectrometric Protein Identification
T. Wehr, Y. Yan, K. McDonald, K. Bala, A. Paulus and N. Liu
Bio-Rad Laboratories, Life Science Group, 6000 James Watson Drive, Hercules, CA 94547, USA
Introduction
Gel electrophoresis is considered to be a labor
intensive and cumbersome separation method. To
visualize proteins after the mass separation,
staining is necessary, adding to the time and labor
requirements. We are introducing Criterion Stain
Free gels containing unique components in its
Tris-HCl gel solution which provide fluorescent
detection of proteins within 5 min after
electrophoresis separation, such shortening the
workflow and reducing the hands-on time of gel
electrophoresis. The unique compounds react with
tryptophans in an UV light-induced reaction to form
fluorescent products in the visible range and thus
allowing protein band detection. The modification
of the tryptophan residues raises concerns
regarding the compatibility of this method with
follow-on methods such as western blotting and
LC-MS/MS or MALDI-TOF MS protein
identification.
Methods
HeLa cell total proteins were extracted from
cultures in a urea-containing lysis buffer. The total
proteins were separated on either 1-dimensional
(1-D) or 2-dimensional (2-D) stain free gels. The 1-
D gels were blotted to nitrocellulose membranes
and probed with a selection of antibodies,
including an antibody to detect the Bio-Rad
WesternC protein standards. The 2-D gels were
stained with Coomassie following UV light imaging.
Protein spots with different intensities were
randomly selected from the gels for protein
identification by mass spectrometry using either
LC-MS/MS or MALDI-TOF MS. The HeLa total
protein separated on the normal Criterion Tris-HCl
gels served as control.
Results
The stain-free gels exhibited no difference to the
normal gels in western blotting using tested
antibodies. The WesternC protein standards, LIM
kinases were detected with similar patterns and
intensities on the stain free and control gels. For
MS protein identification, the same set of high
abundance proteins selected from the stain-free
and control 2-D gels returned the same identities
whether by LC-MS/MS or MALDI-TOF MS, even
through some tryptophan residue-containing
peptides were often missing in the peptide
coverage for the stain free gel samples. Database
searching with a dynamic modification of the
tryptophan-residue recovered a few but not all
tryptophan-containing peptides. However, this
search modification might help identifying the low
abundance proteins on the stain free gels.
Innovative aspects
o Novel protein visualization method with no
need for time and labor intensive staining
protocol
o Compatibility of protein visualization with
follow-up applications such as western
blotting, LC-MS/MS and MALDI-TOF MS
analysis
 Proteomics Based on Peptide Fractionation by SDS-Free PAGE.
1 1 1 1 1
Yassel Ramos , Elain Gutierrez , Yoan Machado , Aniel Sánchez , Lila Castellanos-Serra , Luis Javier
1 2 3 1 1
González , Jorge Fernández-de-Cossio , Yasset Pérez-Riverol , Lázaro Betancourt , Jeovanis Gil , Gabriel
1 1 1 2 3
Padrón and Vladimir Besada . Dept. Proteomics, Dept. Informatics, Dept. Bioinformatics, Centre for
Genetic Engineering and Biotechnology, Havana, Cuba

Introduction SDS-PAGE), allowed the identification of 97

In the absence of SDS, the driving force for the proteins, including low-abundance components.
electrophoretic migration of peptides toward the
anode is supplied by negatively charged amino Innovative aspects
acids and other modifying groups such as • We developed a straightforward method highly
phosphate, sulfate and sialic acid, while the convenient for hydrophobic proteins that links
resulting mobility depends on both the charge and protein separation (in SDS) with peptide
the molecular mass of the peptides. Here we fractionation (SDS-free).
demonstrate the usefulness of peptide • The method selects surrogated peptides which
fractionation by SDS-free polyacrylamide gel increase by 2.5-3 times the number of
electrophoresis and its applicability to proteomics identified proteins.
studies. • Negatively charged modifying groups in tryptic
peptides (i.e. phosphopeptides) migrate in the
Methods fastest fraction.
The method combines 1) fractionation of complex
protein samples by SDS-PAGE in several bands References
1
that is followed by enzymatic digestion , 2) (1) W. Simpson, R. J., Connolly, L. M., Eddes, J. S.,
fractionation of peptide digests in SDS-free Pereira, J. J., Moritz, R. L., and Reid, G. E.
PAGels, and 3) peptide separation and (2000) Electrophoresis 21, 1707-1732.
identification by conventional RP-LC MS/MS. The
first separation benefits from the enhanced
solubility of proteins in the presence of SDS. In the
second step peptides (derived from a defined
molecular mass range of proteins) are fractionated
along the electrophoretic run according to their
charge to mass ratio. The high resolving power
achieved is due to the combination of three
orthogonal principles of separation.

Results
A straightforward method was achieved for SDS-
PAGE of proteins, enzyme digestion, peptide
transfer and fractionation by SDS-free PAGE,
which was named dual-fractionation
polyacrylamide gel electrophoresis (DF-PAGE).
This method increases the number of identified
proteins 2.5-3 fold with respect to the proteins
identified after direct analysis, and more than 80%
of assigned peptides were found in unique SDS-
free gel slices. A vast majority of identified
peptides (93%) have pI values below 7.0 and 7%
have pI values between 7.0 and 7.35. Peptide
digests that were derived from complex protein
mixtures were in consequence simplified as
peptides that are positively charged are not
recovered in the present conditions. Fractionation
of tryptic peptides from casein allowed the isolation
of phosphopeptides in the fraction of highest Figure 1. Tryptic digestion of the recombinant
migration. The use of SDS for protein fractionation Streptokinase fractionated by SDS-free PAGE. A) Non-
allows analysis of highly hydrophobic proteins and fractionated digest, B-I) Lowest to highest migration
minimal protein loses. The analysis of a membrane fractions.
protein extract from Neisseria meningitides by this
approach, where a very few components represent
about 90% of the total protein mass (estimated by
 External gradient chromatofocusing for quantitative analysis of complex lysates

Karl Burgess, Kit-Yee Tan, Lachislav Tsonev, Terry Sheehan, Ken Cook and Andrew R Pitt
Functional Genomics Facility, University of Glasgow,
G12 8QQ, Glasgow, Scotland

Introduction Innovative aspects

Chromatofocusing is an increasingly commonly
used technique for separation of intact proteins in
complex mixtures. It is usually performed with the
use of ampholytic buffers applied to weak anion
exchange columns for separation of proteins and
peptides in a relatively range of pIs. However, pH
gradients generated are generally poorly
reproducible, narrow ranged and non-linear. We
have applied the pISEP reagents
(CryoBioPhysica) to separation of intact proteins,
including simple standards for methods
development, to complex parasitic lysates and
SILAC labeled mammalian lysates.
• Improved separation characteristics for liquid
phase separation by isoelectric point.
• Comparison of external gradient
chromatofocusing to common proteomics
methodologies.
• Quantitative comparison of treated and
untreated mammalian cell lines.
References
(1) Liu and Anderson, Chromatofocusing high-
performance liquid chromatography: 1.
Practical aspects; J. of Chromatography A.
762, 207-217.

Methods

Methods were developed using SAX and SCX

columns with chromatofocussing using the pISEP
reagents as an intact protein first dimension.
Second dimensions were performed on intact
proteins using monolithic PS-DVB columns, or on
digested peptides using extended-length C18
columns. Complex mixture analysis was performed
using the technique in a genomic validation
experiment on Leishmania donovani lysates.
Finally, multiple biological replicates of SILAC-
labelled mammalian lysates were separated by
chromatofocusing at high resolution followed by
fraction collection, digestion and analysis using
high resolution LC-ESI-ToF MS.

Results

Formation of pH gradients was assessed using

multiple replicates, and comparisons were made of
optimal gradient, column and HPLC system
conditions. Descending pH gradients provided the
most detailed, high resolution chromatograms, with
ascending gradients producing equally high
resolution chromatography, but fewer observed
species, due to acid precipitation of proteins. pH
range of the separation was good (from 9.7 to 2.4
in descending gradients and from 2.4 to 10.75 in
ascending gradients) and linearity of gradients was
exceptional, both with standard and software-
optimised gradient conditions. When applied to
complex lysates, a complete two dimensional
separation and analysis resulted in 1172 unique
proteins were identified in the genomic validation
experiment. Finally, analysis of wild type and
treated cells using the same methodology provides
insights into biochemical pathways.
 Devil in IEF: using electric conductivity to monitor salt concentration of 2-DE
samples

Tzi-Ning Chen, Szu-Yu Wu, Ching-Yu Lin and Han-Min Chen

Department of Life-Science, Institutes of Applied Science and Engineering, Catholic Fu-Jen University, Taipei,
Taiwan

Introduction Innovative aspects

In 2-DE experiment, salts have been known to
severely interfere with the result of IEF. To
overcome the interference of salts, desalting
procedures such as protein precipitation or dialysis
are generally utilized but sometimes result in
unwanted lose of proteins. Prior to performing IEF,
accurately monitoring the salt concentration of
samples may be helpful to decide the necessity of
using desalting procedures. In this study, the
portable conductivity meter has been found a cost
effective and accurate apparatus to fulfil this goal.
The influence of salts in IEF experiment was also
reassessed.
• Development of a convenient and accurate
mean to measure salt concentration of 2-DE
samples.
• Characterization of the influence limit of salts
and corresponding remedies for IEF procedure.
• New look of IEF program in focusing proteins.
References
(1) Heppelmann C.J. et al, A simple method to
remove contaminating salt from IPG strips
prior to IEF; Electrophoresis. 2007,
Nov;28(21):3988-91
NaCl standard curve PBS standard curve
14000 14000

A 12000
B 12000
C
Methods 10000 10000

A portable electric conductivity meter was used to 8000 8000

μS

μS
monitor the salt concentration of samples. The salt 6000 6000

4000

concentration of samples was measured by

4000

2000
‧ E.coli crude extract ‧ E.coli crude extract
2000
‧ Pig liver crude extract ‧ Pig liver crude extract
calibrating the observing conductivity (μS) against 0
0
‧ Chinese Pennisetum crude extract
100 200 300 400
0
0.0 0.2
‧ Chinese Pennisetum crude extract
0.4 0.6 0.8 1.0 1.2

standard curves for various salts, such as NaCl NaCl concentratrion (mM)

0 20 40
PBS dilute ratio

60 80 100 120
27.4 54.8 82.2 109.6 137 164.2

and phosphate (Fig 1). To evaluate the influence 0 10

NaCl concentration(mM)

20 30 40 50 60
2.02 4.04 6.06 8.08 10.1 12.12

of salts in IEF experiment, protein lysates prepared Na2HPO4 concentration(mM)

from swine liver were separated using 7cm IPG
strips (pH 3-10) with different focusing programs.
The electrophoretic result of the second dimension
12.5% SDS-PAGE gels were revealed using
VisPro 5 minute protein stain. The focusing results
of IEF were investigated for 2-DE samples
containing exogenous supplement of NaCl from 0
to 120 mM.
Figure 1. Utilization of conductivity to measure the salt
concentration of 2-DE samples. (A) Conductivity meter
(B) The relationship between the conductivity (mS) and
the NaCl concentration (mM) in 2-DE sample buffer. (C)
The relationship between the conductivity (mS) and the
serially diluted PBS in 2-DE sample buffer. The salt
content in three TCA precipitated 2-DE samples (E. Coli,
pig liver and Chinese Pennisetum) were calibrated
against two standard curves respectively.

Results 30750Vhr 20750Vhr 5750Vhr 3750Vhr 2750Vhr

We have developed a convenient procedure of

using a portable conductivity meter to monitor the 6000
V
uA
Over focusing
120 6000 120 6000 120 6000 120 6000 120

salt concentration of in 2-DE samples. By

5000 100 5000 100 5000 100 5000 100 5000 100

4000 80 4000 80 4000 80 4000 80 4000 80

Current (μA)

Current (μA)
Voltage (V)

Voltage (V)

A)μ

A)μ

A)μ

3000 60 3000 60 3000 60 3000 60 3000 60

measuring the electric conductivity, the salt 2000 40

Voltage (V)

Voltage (V)

Voltage (V)

2000 40 2000 40 2000 40 2000 40

Current (

Current (

Current (

1000 20 1000 20 1000 20 1000 20 1000 20

concentration of a 2-DE sample can be accurately 0

0

1750Vhr
100 200
Time (minute)
300 400
0 0
0

1250Vhr
100 200
Time (minute)
300 400
0 0
0 100

750Vhr
200 300
Time (minute)
400
0 0
0

550Vhr
100 200 300
Time (minute)
400
0 0
0

350Vhr
100 200 300
Time (minute)
400
0

estimated. When evaluating the influence of salts

in IEF experiments, it was found that up to 15 mM Under focusing Under focusing

of simple salt, such as NaCl, in a 2-DE sample 6000

5000
120

100
6000

5000
120

100
6000

5000
120

100
6000

5000
120

100
6000

5000
120

100

may not affect the focusing result for a 7cm IPG

4000 80 4000 80 4000 80 4000 80 4000 80
Current (μA)

Current (μA)
Current (μA)

Current (μA)

Current (μA)
Voltage (V)

Voltage (V)
Voltage (V)

Voltage (V)

Voltage (V)

3000 60 3000 60 3000 60 3000 60 3000 60

2000 40 2000 40 2000 40 2000 40 2000 40

strip. The electrophoretic desalting procedure may 1000

0
0 100 200 300 400
20

0
1000

0
0 100 200 300 400
20

0
1000

0
0 100 200 300 400
20

0
1000

0
0 100 200 300 400
20

0
1000

0
0 100 200 300 400
20

only remedy the influence of salt at a modest

Time (minute) Time (minute) time (minute) Time (minute) Time (minute)

concentration. A pre-washing procedure of the
sample rehydrated IPG strip is a more effective
mean to diminish the interference of salts.
Additionally, many suggested IEF programs were
found more than sufficient for focusing proteins. In
a 7cm IPG strip, as low as a total 750 Vhour was
required for a complete focusing of 50 μg proteins
(Fig 2). Applying high voltage may not improve the
focusing result but bring in the risk of strip burning.
Figure 2. Evaluation the optimal IEF program for a
regular 2-DE sample. 50 μg of TCA precipitated proteins
from pig livers was separated by IEF (7cm IPG strip, pH
3-10) and 12.5% SDS-PAGE. The IEF programs were
evaluated from total 350 Vhour (Vhr) to 30750 Vhour.
The IEF program with a total 5750 Vhr is the
recommended by the manufacturer, Amersham
Bioscience. A minimal 1250 Vhr was found sufficient to
focus most proteins in the evaluated samples.
 Isoelectric Western Blotting (iWB) after Digital Proteome Chip (dPC) Focusing
† † † † † †
Thomas Miller, James R. Dasch, Malcolm G Pluskal, Russell Garlick, Stephen Haralampu, Bill Skea,
‡
*Bhanu Singh, *David Malarkey and Sun W. Tam
†
Protein Forest, Inc. 100 Beaver St., Waltham, MA, USA; *National Institute of Environmental Health
‡
Sciences; 111 Alexander Drive, Research Triangle Park, North Carolina and Proteomic Fractionation Group,
University of Massachusetts Medical School, 222 Maple Avenue, Shrewsbury, MA, USA

Introduction voltage. The use of a specialized PSQ (Millipore)

Protein Forest has developed a rapid parallel
isoelectric focusing method based on a chip
technology. The digital proteome chip (dPC™) has
41 gel features each present at a unique pH. In its
typical presentation, this would be a gradient from
4.20-6.20 or pH 6.00-8.00, with each gel feature
separated by 0.05 pH units. Complex proteomic
samples can be separated in 30 to 45 minutes
using this technology. We present a method of
transferring the contents of the dPC to PVDF
membranes to allow the immunodetection of the
contents of the dPC, isoelectric western blotting
(iWB). Using rat liver lysates, dPC separation
followed by iWB was used to fractionate and
detect protein isoelectric point changes. Using this
strategy, it is possible to detect potential post-
translational modifications (PTM) on a number of
examined proteins, including fructose 1,6
bisphosphatase (F1,6 BPase) and HSP 90.
second membrane collected protein that had
passed through the primary membrane in both
transfer systems.
The efficient transfer of protein from the dPC to
PVDF membrane has been demonstrated using
mammalian cell lysates (see Figure 2). Human
plasma and E.coli lysates have also been used
(data not presented).
Innovative aspects
• dPC followed by iWB allows rapid
isoelectric separation and detection of
proteins
• Potential PTM can be observed after the
iWB

Methods
dPC were run under standard conditions. For this
analysis both pH 4.20-6.20 and pH 6.00-8.00 dPC
were used. Protein lysate extracted from rat livers
was obtained from NIEHS. To electroelute
proteins from dPC onto membranes, two methods Figure1. Reproducibility of dPC Western Analysis. 0.05
were developed, a wet tank transfer system and a μg biotinylated ovalbumin was fractionated on pH 4.20
semi-dry transfer system. For the tank transfer to 6.20 dPC. The ovalbumin was transferred to PVDF
process, the PVDF membrane is adhered to the membrane using the tank method. After blocking, the
dPC at high pressure using linear polyacrylamide blot was probed with HRP-strepavidin and ECL
as a reversible adhesive. The PVDF-dPC substrate.
sandwich is placed back into the specialized
running chamber containing transfer buffer and the
proteins are electroeluted from the dPC onto the
membrane. For the semi-dry method, a specialized
chamber was developed, wherein the dPC is
placed next to a piece of PVDF membrane. This
sandwich is placed directly between two plate
electrodes. Thereafter the proteins are transferred
from the dPC onto the membrane. Good contact
between the electrodes, dPC, and the PVDF Figure2. dPC Western Analysis of F1,6 BPase from a
membrane is maintained by using several sheets liver lysate fractionated on two pH 4.20 to 6.20 dPC. The
of pre-wetted filter paper on both the cathode and lysates was transferred to PVDF membrane using the
anode sides. semi-dry method. After blocking, the blot was probed
with a F1,6 BPase antibody and followed with a anti-Ig
Results HRP conjugate. Blots were visualized using ECL
Biotinylated ovalbumin was used as a test chemiluminescent substrate.
compound to work out elution conditions for
proteins from the dPC. Parameters examined
included time, voltage and current, in addition to
the need for pre-equilibration in a buffer containing
SDS. It was found that efficient transfer of proteins
to the membranes in both systems was
accomplished in less than 20 minutes at low
 Fast targeted Proteomics on a chip
1 2 1
Zuzana Demianova , Sami Franssila and Marc Baumann
1
Protein Chemistry Unit, Institute of Biomedicine, University of Helsinki, 00014 Helsinki, Finland
2
Micro and Nanosciences Laboratory, Helsinki University of Technology, 02015 Espoo, Finland

Introduction between Alzheimer patients and control were

One- and two-dimensional polyacrylamide gel measured by 2-DE immunoblotting (Figure 2C).
electrophoresis (1-DE and 2-DE) is a biochemical In conclusion, these chips allow fast, reliable and
method which is capable of separating proteins by sensitive analyses, and a possibility for biomarker
their mass (1-DE) or by their charge and mass (2- screening.
DE). It is one of the most powerful protein
separation methods. However, the current devices
are slow and laborious to use. Our goal was to A B
design miniaturized one- and two-dimensional slab
gel devices (1D-PASGE- and 2D-ComPress-chip)
for a fast targeted proteomics with possibility to
automate and avoiding the manual workflow and
cross contamination.

Methods
Our PASGE- and ComPress-chip are based on the
classical technology including the polyacrylamide Figure 1 → Photographic illustration of miniaturized
instruments. A) PASGE-chip and B) ComPress-chip
matrixes. The PASGE-chip (3x2.5x1 cm, width x
length x depth) is capable of running five different
samples. The ComPress-chip (4x5.2x2.2 cm) is A kD HbA2
A
1 2 C
3 HbF HbA1c
able to perform a 2-DE in a single analysis. The HbS
chips separated a set of predefined standards as HbA

well as tissue homogenates. The gels were 250

stained with Coomassie blue or silver stain, or 150 B

100

used for immunodetection visualized by ECL plus. 75

50 ctrl
Standard proteins were in-gel alkylated and 37

digested with trypsin. Peptide digests were 25

20

analyzed by MALDI-TOF-MS and peptide mass 15 AD

fingerprints (PMF) compared with data available in
database. These results were compared to those
achieved with the regular-sized instrument.
Results
1D-PASGE-chip
The 1D-PASGE-chip separated proteins within 11
min with the gel-to-gel repeatability of 3.8%. The
bands had 5-times higher sensitivity due to the
extremely low-diffusion of the protein bands. The
PMF analysis showed improved peak intensity of
the peptide fragments with considerably lower
enzyme consumption. Additionally, an immunoblot
was used to detect α-actin in the vascular smooth
muscle cell sample (540 ng of total protein) with a
mouse monoclonal antibody (Figure 2A).
2D-ComPress-chip
The ComPress-chip presents a novel way of
combining the first- and second dimension-
separations by pressing. The performance was
checked using a set of known proteins focused in
pH gradient 3-10 and separated in ~ 80 min.
Native 2-DE with ultra narrow pH gradient (pH 6.7-
7.7) was applied to separate HbA1c from other
haemoglobin variants (Figure 2B). The changes in
the post-translational (phosphorylation and
glycosylation) modification of GFAP isoforms
C
Figure 2 → Performance of a 1D and 2D miniaturized
instruments. A) Separation of 540 ng of smooth muscle
cell lysate: line 1| Coomassie blue staining of whole
lysate by PASGE-chip, immunoblotted α-actin band
runs by line 2| the PASGE-chip and by line 3| the mini-
PROTEAN 3 cell; B) native 2-DE of haemoglobin
variants from human blood (Coomassie blue); C) 2-D
maps of post-translation modification differences of
GFAP protein isoforms between control and Alzheimer
disease patient from the frontal cortex of human brain
(Immunodetection)
Innovative aspects
• PASGE- and ComPress-chip are easy-to-use
miniaturized instruments
• Comparison between results obtained from the
PASGE-chip and the mini-PROTEAN 3 cell
show improvements in separation time,
sample and reagent consumption, sensitivity
and repeatability of the chip-based device
• ComPress-chip performs the 2-DE in a single-
step analysis within 80 min
 An evaluation of the suitability of 1.9um particle packed columns for
proteomics applications

C. Blythe, M. Dolci, D. Milton

Thermo Fisher Scientific, Runcorn, Cheshire, WA7 1PR, UK

Introduction • PicoFrits, where a nanospray

The use of sub 2um particles in liquid emitter is incorporated at the end
chromatography has shown many of the column. This eliminates
advantages over traditional 5um packing band broadening caused by dead
materials. To date, however, sub 2um volume between the column and
particle packed columns have been the spraying tip.
employed almost exclusively for small Method
molecule metabolomics and The current presentation evaluates the
biotransformation studies, although their performance of Hypersil GOLD 1.9um
application to large molecule proteomics PicoFrit and Integrafrit formats, using a
research is increasing. Of particular protein digest mixture; a comparison of the
interest for proteomics applications is the performance of 1.9 and 5μm packed
increased efficiency provided by sub 2um columns is also carried out.
particles, which facilitates rapid Results
separations with greater resolution, peak Performance parameters measured
capacity and sensitivity. Due to the sample included peak width, peak capacity and
complexity associated with biomarker and peak height (sensitivity). Investigation of
shotgun proteomics, the need for flow rate limits (optimum flow rate for each
increased chromatographic efficiency particle size), pressure and effect of pore
afforded by high resolution small particle size was also undertaken.
LC is highly desirable. Innovative aspects
It is also very important to maximise the • Sub 2um particle packed
sensitivity of the LC-MS system. This can nanobore columns employed for
be achieved by incorporating in the large molecule proteomics
nanobore column hardware the following: analysis
• IntegraFrits, integrated frits at the • Increased LC-MS sensitivity and
head of the column to eliminate throughput.
peak tailing due to extra-column
volume;
Seppro® Fractionation Platform for Enhanced Detection of Biomarkers in Biofluids

1 1 2 1 3 2
Xiangming Fang , Lei Huang , Weijun Qian , Sergey Sikora , Kimimichi Obata , Richard Smith , Wei-Wei
1
Zhang
1
GenWay Biotech, Inc., San Diego, CA, United States
2
Pacific Northwest National Laboratory, Richland, WA, United States
3
PSS Bio Instruments, Inc., Livermore, CA, United States

Introduction The wide dynamic range of protein satisfactory; (3) SepproTip can be reused up to 30
concentration in mammalian biofluids posts a times. The turnaround time of 12 samples per 55
formidable challenge for low abundance biomarker minutes allows large number of samples being
detection using MS or 2DE technologies. To tackle processed without decrease in sample preparation
this challenge, we developed an avian IgY quality. The SepproTip system makes “digging
antibody-based immunoaffinity fractionation faster” possible for meeting the needs of HTP
®
platform, Seppro IgY-14 for removal of top 14 sample preparation.
high-abundance proteins (HAP), SuperMix for
separation of moderate-abundance proteins (MAP) Innovative aspects
from Low-abundance proteins (LAP), and • IgY antibody-based immunoaffinity columns
SuperEnrich for capturing of proteins from tissue enable highly-specific and effective capture of
leakage. An automated SepproTip system for HTP target proteins from complex biofluids.
sample preparation and facilitating biomarker • Seppro® fractionation platform enhance
validation. detection of LAP from top-down or bottom-up
approaches.
Methods To generate plasma protein-based • SepproTip automation system meets the needs
SuperMix IgY antibodies, human plasma was first of HTP sample preparation.
depleted of 12 HAP via IgY-12 column. The protein
mixture in flow-through fraction was used as
antigens to immunize chickens and as affinity
ligands to purify antibodies. The antibodies were
covalently coupled to microbeads. To fractionate
plasma samples, an IgY-14 LC10 column was
tandem connected to a SuperMix IgY LC2 column.
The fractions are analyzed by LC/MS/MS. To
generate cell protein-based SuperEnrich IgY
antibodies, proteins isolated from cell lines were
used as antigens and affinity ligands. The
antibodies were covalently coupled to polymeric
microbeads or magnetic beads. Cell lysates and
plasma samples were processed through
SuperEnrich column, and the resulting fractions
were analyzed by LC/MS/MS.
®
Results By coupling Seppro IgY-12 or 14 HAP
removal with the MAP-separating SuperMix
system, proteins less than 1ng/ml in plasma were
detected by LC/MS/MS analysis. The SuperEnrich Figure 1. Enhanced detection of LAP in plasma using
IgY columns against prostate cancer, lung cancer, IgY-12 coupled with SuperMix columns. The results
and lymphocyte cells were shown to be able to show more than 2 fold increase of protein identification.
capture more than 500 cellular proteins in each
cell line. Cellular proteins, such as membrane,
nuclear, and mitochondria proteins, were captured
in plasma samples, indicating the usefulness of the
columns for tissue leakage biomarker enrichment
from plasma or serum samples. The SepproTip-
treated samples were analyzed by 1DE, 2DE, and
MALDI-TOF-MS. The results demonstrated that
(1) SepproTip specifically removed all target
proteins; (2) the reproducibility of SepproTip is
 A multi-approach experiment to analyse aphid salivary proteome
1 1 2 1
Nicolas Harmel , Eric Haubruge , Edwin De Pauw , and Frédéric Francis
1
Gembloux Agricultural University, Functional and Evolutionary Entomology, Passage des Déportés
2, 5030 Gembloux, Belgium
2
University of Liege, Mass Spectrometry Laboratory, Sart Tilman, B22 building, 4000 Liege, Belgium

Introduction some inadequate interference during the first

Aphids (Insecta ; Hemiptera) constitute one of
the most important group of agricultural pests.
The role of aphid saliva in the first contact
between the insect and the plant is crucial
during feeding step. It is known that this matrix
contains proteins that prevent plant defence
reactions allowing aphids to ingest phloem sap
continuously for many hours or even days from
a single sieve element. That is why we
investigate aphid saliva proteome in search of
some proteins eliciting plant defence reactions.
Methods
A multi-approach experiment based on the use
of an optimal saliva collection methodology
and both in-solution and in-gel protein
digestion associated to complementary mass
spectrometry techniques was performed in
order to investigate the saliva proteome of an
aphid species (Fig 1). Myzus persicae aphid
salivary proteins were collected with artificial
diets and were either directly in-solution
dimension migration in the 2D electrophoresis.
This experiment led to identification of 5
candidate proteins (glucose oxidase, glucose
dehydrogenase, NADH dehydrogenase, α-
amylase and α-glucosidase) implied in plant
defence manipulation.
Innovative aspects
• First investigation of an aphid (an
organism with a non-sequenced genome)
saliva proteome
• Constitution of a database containing
hundred of aphid (public and home-made)
nucleotides
References
(1) N. Harmel et al, Diversity of aphid salivary
proteins: a proteomic investigation on Myzus
persicae; Insect Mol. Biol. 2008 (in press)
Parafilm

digested or were separated by 2D SDS-PAGE Diet

before trypsin digestion. Resulting peptides

were then identified by mass spectrometry
PVC tube
(LC-MS/MS and MALDI-TOF-MS/MS) coupled After 2 days

with data bank investigations constituted of

expressed sequence tags (EST) from the pea Aphids

aphid Acyrtosiphon pisum and the peach aphid

Soluble saliva Rinsing of the lower
Myzus persicae. The enlarged sequences collection Parafilm layer for
solid saliva collection
were submitted to the BLAST procedure to
identify peptide functions (1).
2D-PAGE
Results (IEF, SDS)

In the process of identifying the salivary IN-SOLUTION

DIGESTION
proteins from M. persicae, the use of specific
EST aphid databases led to the lengthening of IN-GEL
DIGESTION

the peptide sequences obtained from mass

spectrometry. From sequences of only a few
amino acids, sequences of hundreds of LC-MS/MS MALDI-TOF-MS/MS
corresponding nucleotides were obtained using
the EST genomic databases after translation
and adaptation to mass spectrometry data PEPTIDES IDENTIFICATION
(according to specific EST databases)
requirements. It is important to note that the
identification of many aphid salivary proteins
was possible only due to the use of the specific BLAST
homemade aphid EST databases.
In-solution digestion was better adapted than
in-gel digestion for the study of aphid saliva Figure 1. Strategies for aphid salivary protein
proteins, notably due to the gelling consistency separation and identification.
of the collected saliva, which might cause
 Ultrasonic Assisted Protein Enzymatic Digestion applied to gel-separated proteins

Marco Galésio, Diana Vieira, Raquel Rial-Otero, Carlos Lodeiro, and José L. Capelo
REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica, Portugal

Introduction
The development of rapid methods for protein
separation, identification and quantification has
important clinical and toxicological implications. In
the last decades, continuous progress in analytical
methodology has been achieved. New findings in
sample treatment based on ultrasonic assisted
protein enzymatic digestion (USAPED) after
polyacrylamide gel electrophoresis separation
have been developed in our laboratory [1]. The
aim of this work was to compare the overnight and
the new ultrasonic methodologies for protein
digestion when fluorescent dyes are used for
protein visualization.

Methods
For the purpose of this study, a standard protein
mixture of glycogen phosphorylase b, 97 kDa;
bovine serum albumin, BSA, 66 kDa; ovalbumin,
45 kDa; carbonic anhydrase, 30 kDa; trypsin
inhibitor, 20.1 kDa; and α-lactalbumin, 14.4 kDa,
was used.
Stain solutions were prepared diluting the stock
solutions of Sypro Red and Sypro Orange 1:5000
in acetic acid (7.5%, v/v). Protein bands were
observed in an electronic UV transiluminator
radiating at 300 nm. Ultrasonic protein enzymatic
digestion was performed in a sonoreactor system.
Protein identification after sample treatment was Figure 1. Comparison of the overnight protocol (orange
done through Peptide Mass Fingerprint (PMF) color) and the ultrasonic sample treatment with 4 min of
using a MALDI-TOF-MS system model Voyager digestion time (green color) in terms of protein sequence
DE-PRO Biospectrometry Workstation equipped coverage (%) and number of peptides matched. The
with a nitrogen laser radiating at 337 nm. Sypro Red staining was used.

Results Innovative aspects

The accelerated procedure of in-gel protein
digestion was performed in a sonoreactor
operating at 50 % amplitude according to the
method previously developed in our laboratory for
coomassie blue stain [2]. For enzymatic digestion,
sonication times of 2 and 4 min were assayed.
All proteins were identified positively for a total
amount of in-gel protein comprised between 0.8
and 1.8 μg. When the sonication time was 2 min
the protein sequence coverage (%) and the
number of peptides matched were lower than
those obtained with the overnight sample
treatment. When the sonication time was
increased to 4 min, similar results were obtained
for the overnight and the ultrasonic procedures in
terms of protein sequence coverage (%) and the
number of peptides matched. Only minimal
differences were observed for phosphorilase b and
α-Lactalbumin, for which slightly better results
were obtained with the ultrasonic protocol.
• Application of the USAPED procedure to
fluorescent dyes.
• Reduction of protein digestion time from
approximately 12 hours (overnight) to 4
minutes (USAPED).
References
(1) R. Rial-Otero et al. Sonoreactor-based
technology for fast high-throughput proteolytic
digestion of proteins. J. Proteome Res. 6
(2007) 909-912.
(2) F.M. Cordeiro et al., Simplifying sample
handling for protein identification by peptide
mass fingerprint using matrix-assisted laser
desorption/ionization time-of-flight mass
spectrometry Rapid Commun. Mass Spectrom.
21 (2007) 3269–3278.
 Analysis of active proteins extracted from deer antler by 2D-SEC-IEC

Liang Gao, Zhen Liang, Lihua Zhang, Yushu Huo, Yukui Zhang
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China

Introduction
Deer antler is a well-known traditional Chinese
medicines (TCMs), with the obvious regulation
effects on diseases of nervous system, Innovative aspects
immunologic system, cardiovascular system and • Research on the bioactive components in deer
genital system. The bioactivity of deer antler has antler
been considered to be related with the • Two dimensional method containing SEC and
components, such as peptides and proteins. In IEC for high resolution protein separation with
order to investigate the molecular mechanisms of bioactivity maintained
the regulation effects of deer antler, the separation
of proteins in deer antler was performed with off-
line two dimensional size-exclusion 1000 f1 f2 f3 f4 f5
chromatography (SEC) and ion-exchange
chromatography (IEC), to keep the protein activity, 800

so that further screening of bioactive components

could be performed. mV 600

Methods 400

The deer antlers were frozen by liquid N2 and

ground into small particles. Then the proteins were 200

℃
extracted by 50mM Tris-HCl, pH 7.5, at 4 for 72
0
hours. The extracted proteins were firstly
separated on a G3000SW XL column (Tosoh, 0 5 10 15 20

Japan) by molecular weights. Subsequently, the min

collected fractions were concentrated to 1/10th-

volume ratio of original with rotary vacuum Figure 1. Separation of proteins in deer antler by SEC
evaporator and further separated on a SuperQ-
5PW column (Tosoh, Japan) based on the
isoelectric points.

Results
In order to study the bioactive components of deer
antler, the complicated extracted proteins should
mV

be separated into fractions containing as few

protein as possible. Therefore, an off-line two
0
dimensional HPLC method with SEC and IEC
st nd
respectively as the 1 and 2 dimension
separation modes were chosen, so that the
proteins could not be denatured and the bioactivity 5 10 15 20 25

could be maintained after separation. From min

Figures 1 and 2, it could be seen that the coeluted

peaks in the fractions from the SEC column could Figure 2. Separation of the third fraction from SEC by
be further separated by IEC due to the improved IEC
resolving power and different retention
mechanisms. With such a bioactive kept platform,
a peak capacity of about 120 was achieved. The
improved separation capacity of 2D-HPLC is of
great help for the further screening of target
components.
A novel method is described for detection of trace impurities present in purified
biopharmaceuticals associating proteomics studies with hexapeptide ligand libraries. Under
defined conditions the latter largely enhances the concentration of protein impurities to
detectable levels. Then the resulting sample is submitted to proteomics analysis involving
two-dimensional electrophoresis and mass spectrometry with the aim to detect and identify
intact foreign proteins as well as their fragments. Several examples will be shown among
them recombinant Protein A, expressed in Escherichia coli and supplied as 99% pure,
recombinant human albumin, expressed in Pichia pastoris and certified as 95% pure and
purified monoclonal antibodies. In all cases a number of additional polypeptide chains, not
visible in the control, could be detected and obtained in sufficient amounts for MS analysis. In
the cases of recombinant proteins, it could be demonstrated that a number of these
polypeptide chains were host cell proteins still present in the purified product. In addition, a
substantial number of signals (2-DE and mass spectrometry) were found to be cleavage
products of the original r-DNA species. Treatment with ligand libraries of purified proteins is
thus seen as a very powerful method of capture and concentration of host proteins and
cleaved products for further analysis in order to better control the quality of biotechnology
derived products.
 PHOSPHOPEPTIDES: COUPLING OF
IMMUNOAFFINITY AND TIO2 METAL AFFINITY
1 2,3 2,3 4 1,2,3
François Guillonneau , Lamya Haddaoui , Patrick Mayeux , Claude Netter , Luc Camoin .
1 2
Plateforme Proteomique Université Paris Descartes ; Institut Cochin, Université Paris Descartes, CNRS
3 4
(UMR 8104), Paris, France ; Inserm, U567, Paris, France ; Dionex france

Introduction
Obtention of a subset of modified peptides of
interest from very complex mixtures of proteins is
of challenging interest. Protein phosphorylation / Innovative aspects
dephosphorylation is often considered as an • Synergical combination of two techniques
“on/off” molecular switch for cell signaling and yielding improved selection of phosphorylated
many other cellular processes. However, for MS peptides sequenced by MALDI TOF TOF
based identifications, modified peptides represents MS/MS
only an infinitesimal fraction. Enrichment via • Commercially available chromatography
immunoaffinity has greatly helped and so has columns
metal affinity columns(1). Both techniques have
their flaws, the first yields a cohort of non specific References
peptides, and the second looses selectivity when (1) Pinkse et al, Selective Isolation at the
non phosphorylated peptides are too abundant. Femtomole Level of Phosphopeptides
Here we have combined both techniques to from Proteolytic Digests Using 2D-
increase our confidence in peptide identification NanoLC-ESI-MS/MS and Titanium Oxide
and modifications’ localisation. Precolumns; Anal. Chem.2004, 76,3935-
3943
Methods
Proteins from biological samples were extracted
and digested according to the Phosphoscan® kit
protocol from Cell signalling Technology. After C18
purification and concentration, Phophotyrosine-
containing peptides were immunoprecipitated
using mouse mAb (P-Tyr-100) beads. Eluted
peptides were separated on an Ultimate 3000 2D
nanoLC system using a novel Dionex TiO2
µprecolumn (1cm length, 200µm i.d.) and
fractionated on a MALDI plate for MS/MS
identification through Mascot 2.2. Flow-trough (ft)
and eluted (po) fractions were both collected after
separate reverse phase fractionations.

Results
We have compared the same samples with or
without TiO2 filtering. Although immunoaffinity
yielded many peptides bearing phosphate groups,
those were still among an impressive number of
nonphosphoryated ones. The implementation of
the TiO2 column allowed discrimination between
the two species to an almost perfect yield. Thus
we can argue that this supplemental step is
improving separation and selection of
phosphorylated precursors for MALDI MS/MS.
However we still need to determine the limitations
of the TiO2 microcolumn, since a large excess of
nonphosphorylated peptide lead to a partial loss of
selectivity.
We were also able to make a “shortlist” of
phosphorylated species which are identified in
each fraction regardless of the sample treatment,
in other words a kind of “phosphorylated baseline
signal in human cells”.
 Separation of Membrane Proteins and Protein-Complexes Using Free-Flow
Electrophoresis
1 1 2 1 1
Hartmann K , Kronbauer S , Islinger M , Eckerskorn C , Nissum M
1
BD Diagnostics, Innovation Center Biotechnology, Martinsried, Germany
2
Department of Anatomy and Cell Biology II, University of Heidelberg, Germany

Introduction membrane protein complexes. Hydrophobic

Free-flow electrophoresis provides an alternative
separation methodology to 2D-gel electrophoresis
that is purely liquid based and not limited in the
applicable pH or size range. The obstacles remain
the hydrophobic nature of the membrane proteins
and their tendency to precipitate at their isoelectric
point. A further drawback of gel-based methods is
that they are carried out in a strongly denaturing,
detergent-based environment. Under such
conditions, topological information is lost. To
overcome these limitations we developed a novel
separation mode for FFE called interval zone FFE
(IZ-FFE). In contrast to the isoelectric focusing
mode, the separation is carried out at a constant
pH relying on the net protein charges. The applied
pH of the separation buffers is selected such that it
is different from the isoelectric point of the proteins
to be separated thereby maintaining proteins in
solution which otherwise would precipitate.
OXPHOS complexes were separated in native
form by IZ-FFE as demonstrated by BN-PAGE of
individual fractions. Thus enabling a novel 2D
separation strategy for protein complexes.
Innovative aspects
• FFE provides an alternative method for the
separation of membrane proteins. Protein
precipitation can be avoided by using the IZ-
FFE mode. Coupling to LC-MS/MS is
facilitated by the introduction of a cleavable
detergent in the separation media.
• Furthermore we propose a new 2D native
electrophoretic protocol for separation and
identification of membrane protein complexes.
It combines the use of the novel IZ-FFE mode
followed by BN-PAGE to separate the
complexes first by charge and second by
molecular weight.

Methods
Membrane protein extracts from HeLa cells were
prepared including washing steps with sodium
carbonate buffer and dissolving the final pellet in a
low-salt buffer, containing a proprietary cleavable
detergent. The OXPHOS protein complexes were
solubilized in a low-salt buffer, containing digitonin
as detergent. IZ-FFE was carried out at constant
pH 7.8. For further analysis of separated protein
complexes BN-PAGE was performed. Collected
protein fractions were digested and analyzed using
LC-MS/MS (Agilent 1100 binary HPLC system
coupled to a Bruker HCTultra ion trap mass
spectrometer).

Results
We observed precipitation in the separation
chamber when the membrane protein extract from
HeLa cells was separated in the IEF-FFE mode
even when a detergent was included in the
separation media. Application of the IZ-FFE mode
significantly increased protein solubility and
prevented precipitation. The separation media
containing the cleavable detergent facilitated the
coupling to LC-MS/MS since no cumbersome
detergent removal was necessary. Membrane
proteins identified by LC-MS/MS of the individual
FFE fractions were analyzed based on the number
of transmembrane domains using the TMHMM
software (www.cbs.dtu.dk/services/TMHMM/).
The inner mitochondrial membranes extracted
from rat liver mitochondria were chosen as a
model for setting up analytical methods for
Utilization of Immunoaffinity Interaction for Specific Antibody and Peptide Separation
Md. Fida Hasan1*¶, Yoichi Kumada2§, and Shigeo Katoh1, 2
1
Dept. of Molecular Science and Material Engineering, Graduate School of Science and
Technology, Faculty of Engineering, Kobe University, Japan, 2 Dept. of Chemical Science and
Engineering, Faculty of Engineering, Kobe University, Kobe 657-8501, Japan
Present Address: * ¶ Department of Process Engineering and Applied Science, Food Science
and Technology Program, 1360 Barrington Street, P.O. Box 1000, Halifax, NS, B3J2X4,
Canada, Fida.Hasan@Dal.Ca; Tel: 1-902-494-3211, Fax: 1-902-420-0219
§ Department of Chemistry and Materials Technology, Kyoto Institute of Technology, Kyoto
606-8585, Japan
ABSTRACT: In recent years, immunoaffinity, using methods that are both sensitive and
specific, has challenged classical techniques. The highly specific antibodies constitute a
promising tool in the isolation of functionally homologous peptides from protein hydrolysates.
The separation of antihypertensive fragments from water extracted bonito protein hydrolysates
(WEBPH) has been studied by using antibodies. We produced antipeptide antibodies against
modified C-terminal peptide (KKPTHIKWGD, PC-IACE) from tuna glyceraldehydes-3-
phosphate dehydrogenase (GAPDH). The GAPDHs are a family of novel peptides with strong
antihypertensive properties. This modified peptide was used as a model to demonstrate the
immunoaffinity screening strategy. An efficient procedure for the separation of high
immunoaffinity peptides from a WEBPH with inhibitory activity was developed. Polyclonal
antibodies have been generated by immunization of rabbits with chemically synthesized PC-
IACE conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An antipeptide
antibody was purified against a PC-IACE by CNBr activated Sepharose peptide column
chromatography. Both antibodies were used in competitive enzyme-linked immunosorbent assays
(ELISA) to binding characteristics against peptide-BSA conjugate. The specificity of antibodies
was evaluated by a indirect ELISA and a competitive indirect-ELISA. The ELISA was valuable
for detecting the existence of PC-IACE specific antibodies in the sera and purified products and
quantifies the antibodies. In Dot blot analysis, the purified antibody also recognized a peptide-
BSA conjugate.
This highly specific and sensitive antipeptide antibody provides an important tool for the
separation of peptide drug, with immunoaffinity interaction from the other members of the similar
peptides. Immunoaffinity chromatography columns with the immobilized purified antipeptide on
CNBr activated Sepharose gel retained the homology peptide from the WEBPH. Monitoring the
purification of hydrolyzed peptide with antipeptide antibodies suggests that while the
performance of the evaluated purification procedures would be reasonably acceptable in terms of
their yield, recovery and purity are attractive. Applications of these antibody tools are suggested
for the rapid detection and purification as well as evaluation of specific binding dynamics.
 Solid Phase Extraction - Liquid Chromatography (SPE-LC) Interface for Automated
Peptide Separation and Identification by Tandem Mass Spectrometry
O.B. Hoerning; M.B. Andersen; O. Vorm
Proxeon A/S, Staermosegaardsvej 6, DK-5230, Odense M, Denmark

Introduction
Solid phase extraction (SPE) is a simple, widely
used technique for desalting and concentrating
peptide and protein samples prior to mass
spectrometry analysis. Often, SPE sample
preparation is done manually and the samples
eluted, dried and reconstituted into 96 well titer
plates for subsequent LC-MS/MS analysis. To
reduce the number of sample handling stages and
increase throughput, we developed a robotic
system to interface off-line SPE to LC-ESI-MS/MS.

Method
Samples were manually loaded onto disposable
SPE tips that subsequently were connected in-line
with a capillary reverse phase (RP) column.
Peptides were recovered from the SPE step and
separated on the RP column using isocratic elution
conditions and analyzed by electrospray tandem
mass spectrometry. Flow was delivered by two
nanoflow piston pumps operated with Advanced
Flow Control (AFC) (Proxeon, Denmark). Using a
modified autosampler for mounting and disposal of
the SPE tips, the SPE-LC-MS/MS system could
analyze 8 samples per hour. Up to 96 SPE tips
can be processed in one batch.

Results
The chromatographic performance of the SPE-LC
system was evaluated in terms of peptide ion peak
widths, column peak capacity and retention time
reproducibility based on the analysis of tryptic BSA
and a 12 protein component mixture. Peptide
mixtures eluted within approximately 5 minutes,
with individual peptide peak width of ~5 seconds
(FWHM), making the SPE-LC suited for high
throughput analysis. The MS/MS data was
extracted and searched against a protein database
using the Mascot search engine resulting in
confident identification of the standard proteins.
The relatively high sample throughput, separation
power and high sensitivity makes the automated
SPE-LC MS/MS setup attractive for proteomics
experiments as demonstrated by the identification
of the components of simple protein mixtures and
of proteins recovered from SDS-PAGE and 2DE
gels.

Innovative aspects
Sensitive, high throughput analysis of peptide
samples by a novel SPE-LC - MS/MS set-up.
 2D protein and peptide separation of serum by preparative monolithic
chromatography
1 2 3 1 1
Linda IJsselstijn , Deborah Kronenberg , Remco Swart , Peter J. Koudstaal , Peter A. E. Sillevis Smitt ,
2 1
Monique M. B. Breteler and Theo M. Luider
1 2
Department of Neurology and Department of Epidemiology and Biostatistics, Erasmus Medical Center,
Dr. Molewaterplein 50, 3015 GE Rotterdam, the Netherlands
3
Dionex Benelux B.V., Abberdaan 114, 1046 AA Amsterdam, the Netherlands

Introduction increase in compounds observed by the software

The search for biomarkers in serum is complicated
by the presence of high abundant proteins. 2D
separation using monolithic columns can solve this
problem to a certain extent. The relative short
separation times and the possibility to separate
both proteins and peptides at high-resolution make
this column material optimally suited for the
detection of low abundant proteins and peptides in
complex samples (e.g. serum). The developed
protein separation and subsequent peptide
separation has been compared to 1D peptide
separation of identical samples.
(16000) compared to 1D peptide separation
(6000).
Innovative aspects
• The identification of low abundant proteins in
serum by 2D separation on monolithic columns
• By the 2D approach we could detect about
16000 monoisotopic peaks compared to 6000
by the 1D approach
6
• By the 2D approach a dynamic range of 10 is
reached in serum

Methods References
Serum was diluted 25 times in 0.05% TFA water (1) This work was supported by the
and 1 μl was injected onto a nanoLC system Netherlands Proteomics Centre
(Ultimate 3000). A custom-made monolithic
column with size 500-μm i.d. × 100 mm was used.
A 25 min gradient of 24-48% ACN was applied and
the fractionation time was varied resulting in 24, 48
and 96 fractions, respectively. The fractions were
enzymatically digested. Separation in the second
dimension was done on an identical system with a
monolithic column with size 200-μm i.d. × 50 mm.
A 10 min gradient of 0-48% ACN was applied and
the flow through was spotted on PAC-plates
(Bruker Daltonics). The fractions obtained in the
first dimension were also spotted on PAC-plates
without further separation. Subsequently, mass
Figure 1: Flow chart of the 2D approach. Dashed line
spectra of the spots were obtained by automated
indicates the 1D approach.
MALDI-TOF/TOF (Ultraflex).

Results
The separation of serum proteins on the monolithic
column was reproducible with an average CV of
0.23 ± 0.18% for retention time. The CVs were
calculated using the retention time of the eight
highest peaks in the UV trace of five successive
runs. This high reproducibility opens the possibility
to run the separations in a repetitive way, enriching
lower abundant proteins. Mass spectrometric
analysis of the 24, 48 and 96 digested protein
fractions showed that by increasing the number of
fractions from 24 to 96 twice the amount of Figure 2: UV-traces (214 nm) of the separation in the
compounds were picked by WarpLC software first dimension (protein level) of five successive runs of
(Bruker Daltonics). In addition, a higher number of the same sample on a preparative monolithic column.
fractions resulted in a relatively higher S/N for low Human serum albumin elutes after 15 min and
abundant peptide peaks. Results of the second apolipoprotein A after 22 min.
separation of 48 protein fractions showed a large
 MSRAT, A Proteomic Sample Software Analysis Tool

Oren Kagen, James R. Dasch, Russell Garlick, Bill Skea, Andrew L. Johnson, ∗§Yakov Chudnovsky,
† † † †∗ †
∗§
David M. Sabatini, ∗Eric Spooner, William C. Hahn, Milan G. Chheda and Howard A. Fine
§# §# ‡
† ∗
Protein Forest, Inc. 100 Beaver St., Waltham, MA,02453; Whitehead Institute for Biomedical Research,
Cambridge, MA 02142; Broad Institute, Cambridge, MA 02139; #Department of Medical Oncology, Dana-
§
‡
Farber Cancer Institute, Boston, MA 02115; and Neuro-Oncology Branch, National Cancer Institute, National
Institutes of Health, Bethesda, MD 20892

Introduction assessed by spectral counting, along with many

The output from a typical proteomics experiment
can often include thousands of matched peptides
and hundreds of identified proteins. As an aid to
organization of these complex datasets, the Mass
Spec Results Analysis Tool (MSRAT) has been
developed. MSRAT has been used in conjunction
with Protein Forest’s digital proteome chip (dPC™)
followed by LC/MS/MS. This tool allows the user
to take MS data in the Excel format from MS
searches such as Sequest or the Excel output
from Scaffold and to organize this data in a way
that allows the user to find relationships between
different pH fractions generated by dPC
fractionation. As an example of the utility of
MSRAT, proteomic data generated from a
glioblastoma cell line grown at two differentiation
states are presented.
Methods
proteins whose expression was not altered.
Using MSRAT, several views of the data are
available, organized to sort by proteins or
peptides, quickly exclude classes of proteins such
as those identified by a single peptide match and
keratins. In the peptide view, which allows rapid
assessment of coverage, examination of potential
database redundancies of the called peptides is
possible. Data can be ported to Excel or Explorer
to aid in presentations or further analysis. Venn
diagrams and a bubble plot of spectral count ratios
are added features of the analysis tool.
Innovative aspects
• MSRAT allows rapid sorting of LC/MS/MS
data for expression analysis
• Many proteins were found to be up- or
down-regulated after dPC isoelectric
separation and LC/MS/MS

Tumour-initiating cells (TIC) share functional References

properties, such as self-renewal and capability for
multilineage differentiation, with normal stem cells. (1) Lee, J. et al. Tumor stem cells derived from
The glioblastoma TIC line 0308 (1) undergoes glioblastomas cultured in bFGF and EGF more
forced differentiation under certain culture closely mirror the phenotype and genotype of
conditions. These cells were grown either in NBE primary tumors than do serum-cultured cell lines.
medium (1), which maintains a basal non- Cancer Cell. 2006 May;9(5):391-403
differentiated state, or in DMEM + 10% FCS, which
causes the cells to differentiate. Lysates of cells at
each differentiation state were reduced and
alkylated and desalted into 10 mM Tris, 5 mM
NaCl, 1% Triton X, and 2% octyl beta
glucopyranoside. Duplicate samples of 100 μg of
each lysate were loaded into a dPC running
chamber containing a dPC (pH 4.20-6.20). Pools
of gel features (0.4 pH pools) were trypsinized and
extracted peptides analyzed by LC/MS/MS using a
Thermo LTQ mass spectrometer. Data were
analyzed by using Sequest and MSRAT.

Results

Using MSRAT to sort the data into pH pool specific

protein calls, we found that 457 proteins excluding
keratins were identified in three 0.4 pH pools (> 2
unique peptide minimum). Among the proteins
identified, over 100 proteins were found whose
expression level was either up- or down-regulated
in DMEM + 10% FCS compared to NBE, as
Figure 1. Screen shot of MSRAT with proteins from TIC
line 0308. Data is sorted by fraction, and duplicates are
in adjacent columns. A known neural precursor marker,
nestin, is highlighted in green.
 Proteome-wide analysis of protein synthesis and –degradation in E. coli
1 1,2 4 1 1 2
G. Kramer , M.A. Nessen , R.R. Sprenger , J.W. Back , , L.J. de Koning , J.H. van Maarseveen , M.J.
3 3 1 1
Teixeira de Mattos , K.J. Hellingwerf , L. de Jong and C.G. de Koster
1
Swammerdam Institute for Life Sciences (SILS) - Mass Spectrometry of Biomacromolecules
2
Van ‘t Hoff Institute for Molecular Sciences (HIMS) - Synthetic Organic Chemistry Group
3
Swammerdam Institute for Life Sciences (SILS) - Molecular Microbial Physiology
4
Acedemical Medical Centre (AMC) – Proteomics facility
Universiteit van Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands,
g.kramer@uva.nl

Introduction
Understanding cellular regulation requires insight References
in the relationship between mRNA expression and (1) Kiick et al, Incorporation of azides into
protein expression. Although the availability of the recombinant proteins for chemoselective
complete genome of various species has resulted modification by the Staudinger ligation; Proc
in a shift from the level of a single gene or protein Natl Acad Sci USA 99, 19-24 2002
to genomics and proteomics, analysis of post- (2) Dieterich et al. Selective identification of
transcriptional regulation on a genome-wide scale newly synthesized proteins in mammalian
is still a daunting challenge. For this purpose we cells using bioorthogonal noncanonical amino
are developing a chemical pulse-chase labeling acid tagging (BONCAT) Proc Natl Acad Sci
system. USA 103, 9482-7 (2006)
(3) Back et al, Mild and chemoselective peptide-
Methods bond cleavage of peptides and proteins at
We employ the tolerance of the methionyl-tRNA azido homoalanine Angew.Chem. Int. ed.
synthetase towards the methionine analogue 117, 8160-8164 (2005)
azidohomoalanine (Azhal) to label the E. coli
proteome (1,2). By using a selective and mild
chemical reaction directed against azides (3) in
combination with diagonal chromatography we
separate labelled peptides from unlabeled species,
thereby enabling mass spectrometric identification
of newly synthesized proteins. Quantification of
proteins labelled by a pulse of Azhal will allow
determining both the relative rates of synthesis
and the half lives of proteins on a genome wide
basis.

Results
To look at protein synthesis on a small timescale, figure1: structure of the methionine analogue Azhal
E. coli cells are grown in mineral medium for 15 used to label newly synthesized proteins in E. coli.
minutes in the presence of Azhal to label newly
syntesized proteins. Under the conditions used,
growth rates of E. coli are the same on Azhal or
methionine during the labeling time used.
Separation of the labeled peptides from the
unlabeled species by diagonal chromatography
resulted in more than 500 newly synthesized
proteins identified with a low false discovery rate.
This for the first time allows proteome wide
detection of newly synthesized proteins in
prokaryotes. Comparison of these findings with
micro-array data will provide valuable insights into
the regulation of protein expression at the
translational level and shed new light upon the
regulation of proteins in the bacterial cell.
figure 2: mild chemical cleavage of azhal containing
Innovative aspects peptides by TCEP, inducing either reduction from an
• Proteome wide analysis of protein azide to an amine or cleavage after Azhal resulting in a
peptide with a c-terminal homoserine lactone residue
synthesis rates in prokaryotes.
and a peptide with a normal n-terminus.
• Diagonal chromatography of azide
labeled peptides proteome wide.
SDR HyperD® Resin for Detergent Removal Prior to Mass
Spectrometry Analysis
Hongshan Li, Saurabh Nagpal, Gurpreet Kaur, Brian Miller and Lisa Bradbury; Pall Corporation

Biological detergents frequently play a critical role in the preparation of protein containing
samples, most commonly for protein solubilization, denaturation, and/or stabilization.
Unfortunately detergents often interfere with downstream applications and analytical techniques,
e.g., mass spectrometry (MS) analysis, thus requiring detergent removal prior to use. There are
many methods for detergent removal including dialysis, gel filtration and ultrafiltration. In this
study, Pall SDR HyperD® resin is evaluated for rapid, efficient detergent removal from protein
samples using Nanosep® spin device, 96-well plate, and 1ml pre-packed column formats. The MS
experimental data shows that SDR HyperD® treatment significantly improves the protein signal
quality and intensity after detergent removal. The sensitivity of MS protein detection increases
more than 20-fold (BSA in the presence of CHAPS, Nonidet P40 or Triton X-100) after SDR
HyperD® treatment. Additionally, high protein recovery (>99% BSA recovery with Triton X-100
removal), efficient detergent capture, and removal of nonionic, cationic, and anionic detergents is
demonstrated. Based on results from this highly sensitive MS based performance
characterization approach, the rapid and efficient detergent removal by SDR HyperD® resin
shows a great deal of potential for use prior to detergent sensitive applications and analyses.
Additionally, the flexibility in choice of format, accommodating low to high throughput and small to
large fluid volumes, makes this resin an ideal sample preparation choice for many protein
applications.
 Microchip-based monolithic immobilized pH gradient
isoelectric focusing for protein analysis

Yu Liang, Yongzheng Cong, Zhen Liang, Lihua Zhang*, Yukui Zhang

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian,China

Introduction References
With the development of μ-TAS techniques, CIEF [1] C. Yang, G. Zhu, L. Zhang, W. Zhang, Y.
has been applied on microfluidic chips, and shown Zhang, Electrophoresis, 2004, 25, 1729–1734.
the potentials of rapid protein analysis and further [2] G. Zhu, H. Yuan, P. Zhao,L. Zhang, Z. Liang,
integration with other operation units or separation W. Zhang, Y. Zhang, Electrophoresis 2006,
modes. Typically, IEF is performed in aqueous 27, 3578–3583.
ampholyte solution. However, carrier ampholytes
(CAs) added in the running buffer might interfere
the following sample detection and even the
multidimensional separation. To overcome such
shortcomings, in our recent work, monoliths with
immobilized pH gradient (M-IPG) was prepared in
the microchannel by UV initiation.

Methods
M-IPG was prepared by photoinitiated
polymerization of acrylamide, glycidylmethacrylate
and N,N-methylenebisacrylamide with 1,4-
butinediol, dodecanol and DMSO as porogens,
within the 100μm-width channels of a glass chip,
followed by the attachment of Ampholine to the Figure1 Photograph of the microchip with monolithic
surface of the porous monolith via epoxide groups immobilized pH gradient (M-IPG) for isoelectric focusing
(Figure 1). To illustrate the performance of such a of proteins
microchip-based M-IPG IEF, several FITC-labeled
proteins were isoelectric focused and concentrated
with monoliths in the channel of glass chip. The
35
focused zones were subsequently mobilized by
replacing NaOH (catholyte) with H3PO4 (anolyte), 30
and detected by a LIF detector.
25

Results
mv

20
With M-IPG materials in the microchannel, a
mixture of 3 proteins was enriched and separated 15

within 1 min. The protein mixture could be

enriched and separated in 1 minute (Figure 2). The 10

avoidance of moving CAs in the buffer could 5

decrease Joule heat generated in the channel, 0.0 0.5 1.0 1.5

enabling high electric field applied. In addition, the t(min)

monolithic media in the channel could also reduce

the diffusion and the drift of focused zones during Figure2 Microchip-based IEF separation of myoglobin
mobilization. Further work on the construction of (pI 7.3), β-Lactoglobulin(pI 5.1) and trypsin inhibitor (pI
4.5)
M-IPG CIEF based 2D-CE on microchip is
undergoing to improve the separation capacity of
microfluidic chips.

Innovative aspects
• Photoinitiated polymerization of monoliths with
immobilized pH gradient (M-IPG) on
microfluidic chips
• Microchip-based IEF with M-IPG for protein
analysis
 Depletion of high abundance proteins in serum by

protein imprinted polymers

1 1,2 1 1 1
Jinxing Liu , Qiliang Deng , Lihua Zhang , Zhen Liang , Yukui Zhang
1
National Chromatographic R. &. A. Center, Dalian Institute of Chemical Physics, the Chinese
Academic of Sciences, Dalian, P. R. China, 116023
2
College of science, Tianjin University of Science and Technology, Tianjin, P. R. China, 300222

Introduction
Current research trends are focused on the Innovative aspects
exploration of proteins in serum to gain insights • Selective binding of serum albumin by MIP
into the molecular behavior of diseases through chromatographic column and monolithic
the proteomics. A key technical challenge materials
confronting the comprehensive analysis of proteins • A rapid, inexpensive and simple method for
in serum is the broadly dynamic range of proteins. depleting high abundance proteins
Since high abundance proteins could undermine • Great potential in the selective enrichment of low
the detection of low abundance ones, it is urgent to abundance proteins.
develop rapid, effective and simple methods to
deplete the high abundance proteins. References
(1) Simpson, R. J., Proteins and Proteomics: A
Methods Laboratory Manual, Cold Spring Harbor
In our recent work, two kinds of molecular Laboratory Press, New York 2003
imprinting polymers (MIPs) for the depletion of (2) Shi, H., Tsai, W. B., Garrison, M. D.,
human serum albumin (HSA) were prepared by Ferrari, S., Ratner, B. D., Nature 1999,
molecular imprinting technique. Hydrogels with 398, 593-597
BSA imprinted were synthesized under low
temperature in a chromatographic column, and
worked as the stationary phase to distinguish the
imprinting and non-imprinting proteins. Another
kind of monolithic imprinting material was prepared
with porcine serum albumin (PSA) as the template.
After grinded into particles, they were used as the
packing materials for SPE to selectively remove
HSA in serum.

Results
In selective test, both imprinted hydrogels and 1
grinded monolithic materials showed specific
binding to the template proteins. Although both
MIPs and non-imprinting polymers (NIPs)
monolithic columns were prepared and evaluated
under the same conditions, the former one could
recognize the template protein from a mixture of
proteins, which could not be accomplished by the
latter one (Fig. 1, Fig. 2). In addition, the selective
binding test for MIP and NIP monolithic materials
was carried out, and MIPs adsorbed more
template protein than other competitive proteins
and NIPs adsorbed proteins equally. These results 2
indicated that imprinted materials showed high Chromatogram of Selective recognition of proteins on
affinity and specific recognition for the target MIP column (Fig. 1) and NIP column (Fig. 2)
protein. Since MIPs are less expensive and Column: 4.6×50 mm Mobile phase (A) pH 6.5 10mM
quicker to prepare than biological receptors, such phosphate buffer (B): (A) +2M NaCl; gradient condition:
0-60 min, 6-100% (B).
materials have great potential in the depletion of
high abundance proteins and even in the
enrichment of low abundance proteins in proteome
study.
 Vascular wall profiling addresses deep proteome from minute samples.
Sebastián Mas, Roxana Martínez-Pinna, Fernando Vivanco, and Jesús Egido
Laboratorío de Nefrologia Experimental Y Patología Vascular, Fundación Jiménez Díaz/Universidad
Autonoma de Madrid. Av Reyes Católicos 2, 28040, Madrid, Spain
Introduction
Over the last 6 years 2DE has been the gold
standard for proteomic characterization of with
excellent results in terms of publications.
Particularly in cardiovascular biomarkers
identification, the last proteins incorporated to the
biomarkers validation pipeline had been obtained
by electrophoretic techniques. In highly
heterogeneous samples, homogenization prior to
its analysis masks local variations criticals for our
understanding of these pathologies.
Development of Peptide/protein profiling
techniques to overcome this limitation, by direct
coupling of proteins or peptides obtained “in situ”
from small homogeneous areas within the tissue,
had provide a new set of data completely different
from those obtained in previous studies. The
present research, carried out in human
atherosclerotic plaques, has shown an equivalent
area studied by standard proteomic approach
using silver-stained bidimensional electrophoresis
in parallel with protein profiling of intimal vascular
muscle cells leads to the identification of a
completely different set of proteins, and more
remarkable many low-abundance proteins
potentially involved in the pathophysiological
process.
Methods
Human Carotid endarterectomy samples cell from
the innermost layer were either excised or
digested in situ from thin-slice frozen samples.
Microdissected samples were analysed using a
conventional approach combining two-dimensional
electrophoresis with MALDI-TOF identification,
while in situ digestion sample were extracted using
a 100 um capillar followed by an on-line analysis
using nanoLC-MS/MS. Data from both approaches
were compared under the same searching
conditions.
Results
Using this approach we can identified with
statistical significance up to 16 proteins from a
2
tissue area as low as 10000 μm from a cryostat-
section of 15 μm thickness. A trypsin droplet of
about 100 pl were added in our area of interest
and after 2h incubation, it were aspirated using the
reverse phase capilar. The protein quantity was
estimated on 0.8 μg.
In parallel we compare this direct approach with
those obtained using 2DE gel from vascular
smooth muscle cells excised from the same
sample. Using in this case a 100-fold increase
both quantity and surface dissected.
Just 3 proteins were found in both lists and even
more important some striking proteins as prolargin
and interleukin-25 were found by in-situ digestion
while 2DE only lists abundant structural or
transport proteins.
Further improvements of this approach could lead
in better protein identification in very small areas of
highly heterogeneous samples.
Innovative aspects
• Samples as low as 100 cells can be subjected
to proteomic analysis
• Most of proteins that were identified by
nanoLC-MS/MS are absent in 2GE.
• Some low abundant proteins were identified
with confidence.
 Use of 1ml Prepacked AcroSepTM Q
Ceramic HyperD®F Resin Combined
with Albumin and Antibody Depletion
Results in Significant Improvement in
Protein Detection by 2D Gel
Electrophoresis and Mass
Spectrometry Analysis

AUTHORS
1 2
Masilamani Selladurai Saurabh Nagpal , Chitra
3 4 5
Thangavel , Gurpreet Kaur , Hongshan Li and
6
Lisa Bradbury
1,2,3,4
Proteomics Group, Pall India Pvt. Ltd,
Bangalore, INDIA,
5, 6
Proteomics Group, Pall US Pvt.Ltd. Woburn,
Boston, USA

ABSTRACT

The challenge of complexity reduction prior

to proteomic analysis has pushed forward the
development of an ever-expanding array of
proteomics products and protocols. From a
practical perspective however, it is crucial to
consider the requirement for easy to use,
reproducible methods that have limited possibility
for sample cross contamination. Additionally, the
importance of minimizing non-specific protein loss
means that the complete final procedure should
have as few steps as is reasonably possible. In the
end, most researchers balance simplicity,
reproducibility, depth of proteomic information, and
cost, in designing their final protocols. Pall has
developed a combination protocol for serum or
plasma that exposes the sample to only 2 protein
columns, with no other steps – ligand specific
depletion of human serum albumin and IgG
followed by ion exchange (IEX) fractionation using
stepwise elution. This combination is very cost
effective and, as all reagents are disposable, has
no potential for cross-contamination. The
TM
Enchant Multi-Protein Affinity Separation Kit,
which depletes using resin in a Nanosep® spin
filter, takes ~20 minutes. Once depleted, samples
are fractionated using IEX Ceramic HyperD high
capacity resins and pH elution. Plasma samples
processed as described then analyzed by 1
dimensional and 2 dimensional gel electrophoresis
and mass spectrometry analysis to demonstrate
the improvement in protein detection as a result of
this sample-processing scheme.
 Protein quantification and separation before 2D electrophoresis using LFII

1 2 2 1
Dr Judit Nagy , Fiona Pereira Dr Stuart Hassard , Tony Cass ,
1 2
Institute of Biomedical Engineering, Imperial College London, deltaDOT Ltd. London UK

One of the most commonly used protein

separation techniques in proteomics is two
dimensional gel electrophoresis (2DE). Although
2DE is cheap to set up and gives a visual profile
of complex protein mixtures, it has many
drawbacks. It is not quantitative, it can be
laborious, it cannot separate the complete protein
set and the reproducibility is low. All these factors
result in increased sample replicates needed in
the discovery of biologically relevant protein
changes. To avoid running low quality samples on
2D gels and wasting valuable materials and time,
samples are first analysed and quality checked
using deltaDOT’s Peregrine system. Protein
detection is based on Label Free Intrinsic Imaging
(LFII™ ) developed by deltaDOT.

LFII™ is a combination of multi-pixel detectors,

advanced signal processing and microfluidics.
This removes the need for any label in the
detection of biomolecules and small molecules.
The detectors are configured so that separated
molecules traverse the 512 pixels in a time
dependent fashion allowing a unique space-time
correlation to be acquired. Smaller proteins move
relatively quickly and therefore present a steep
slope, while larger proteins, moving slowly,
present a shallower slope. The key is that all of
these lines, even though they have different
angles, meet at one point. It is deltaDOT’s ability
to find this point that allows us to image and
identify unlabelled proteins and peptides.

This work-flow not only allows to speed up and

improve the 2DE gel outputs but also gives the
opportunity to verify and quantitate proteins which
are identified from the 2D gels by mass
spectrometry.
 Profiling of Low Molecular Weight Human Plasma Proteins
2 1 1 1 3
DongGuen Sul , Jong Bok Seo , Hye Sook Kim , Jin Hee Lee , Gun Cho ,
2 1
Sohee Phark Jeongju Seo and Myung Hee Nam
1
Metabolome analysis Team, Korea Basic Science Institute, Seoul 136-701 Korea ,
2
Graduate school of medicine, Korea University, Seoul 136-701 Korea
3
Proteomics team, Korea Basic Science Institute, Daejeon 305-333 Korea

Introduction new biomarkers and drug targets from human

Low molecule weight plasma proteins (LMPP) is plasma.
considered as biologically active biomarkers. Major
methodological consideration for the detection of References
LMPP was the depletion of major high molecule (1) G. S. Omenn et al, Overview of the HUPO
weight plasma proteins (MHPP) which contain Plasma Proteome Project; Proteomics
almost 99% of plasma (1). Chromatographic 2007 25: 3226-3245
depletion of MHPP using various monoclonal (2) R. S Tirumalai et al, Characterization of
antibodies to MHPP has been used in many the low molecular weight human serum
plasma proteomic studies. A simple method to proteome; Mol. Cell. Proteomics 2003,
remove MHPP from serum without concomitant 2:1096-1103
loss of LMPP components has been developed by (3) H. M. Georgiou et al, Proteomic analysis
Tirumalai et al. (2). Their method employs of human plasma: failure of centrifugal
centrifugal ultrafiltration using solvent conditions ultrafiltration to remove albumin and other
that disrupt protein-protein interactions so that high molecular weight proteins;
LMPP components bound to larger species are Proteomics 2001,1:1503-1506.
released and become free to pass through the
molecular weight cutoff membrane (COM). In this Mr M
[kDa]
1 2 3 4 5 6 7 8 9 10

study, we applied various buffer to obtain a 75

50

consistant and higher recovery rate of LMPP from 37

human plasma. 25

20
15

Methods
10
Sample Preparation: Human plasma was obtained
by centrifugation at 1300 g for 10 minutes at 4 ºC.
Protease inhibitor cocktail was added to plasma
samples.
Figure 1. 1-DE-PAGE analysis with different
Preparation of low molecular plasma proteins: buffering reagents in the urea buffer system. Plasma
Three milliliters of human plasma were diluted by was diluted 1:4 by adding different buffering reagents to
adding of 12 ml of various buffer systems. The the urea buffer system, then ultrafiltrated using COM.
diluted sample was then loaded onto a filter Ultrafiltrated samples were concentrated using 3 KDa
membrane, and centrifuged at 3000g. The filtrate membranes and 5 ul aliquots of samples were subjected
was concentrated to 200ul using a cut off to SDS-PAGE. lane 1 and 2: 25mM NH4HCO3 + 20%
membrane (Vivaspin, MWCO 3,000) proteins were ACN (pH 8.2); 3 and 4: 40mM Tris-HCl + 20% ACN (pH
separated by SDS-PAGE or 2-DE. 8.2); 5 and 6: 7M Urea, 2M Thiourea + 20% ACN; 7 and
Protein identification: LMPP were digested with 8: 7M Urea, 2M Thiourea, 25mM NH4HCO3 + 20% ACN
(pH 8.2); 9 and 10: 7M Urea, 2M Thiourea, 40mM Tris-
trypsin and were analyzed by FT-ICR MS.
HCl +20% ACN (pH 8.2). The buffer system containing
7M Urea, 2M Thiourea, 25mM NH4HCO3 +20% ACN
Results (pH 8.2) produced the highest recovery rate of plasma
we used various buffer systems, including urea proteins.
buffer solution to determine which one provided
the highest recovery of LMPP using a 30 KDa of
cut off membrane and found that the buffer system
containing 7M Urea, 2M Thiourea, 25mM
NH4HCO3 +20% ACN (pH 8.2) produced the
highest recovery rate (Fig.1). Total LMPP proteins
prepared by urea buffer system were analyzed by
ESI-LTQ FT MS. Among the total 168 proteins
identified, 99 proteins (59%) were belong to the
under 30 kDa. Although this technology will not be
solved completely current problem of biomarker
Fig.2. Distribution of identified proteins by molecular
discovery using human plasma, we expect that this weight. The 99 proteins were
technology represents an opportunity to discover belong to the under 30 kDa (58.9%)
 Selective purification of azide-containing peptides from complex mixtures
1,2 1 2 1 1
Merel A. Nessen , JaapWillem Back , Jan H. van Maarseveen , Gertjan Kramer , Leo J. de Koning , Luitzen
1 2 1
de Jong , Henk Hiemstra , Chris G. de Koster
1
Mass Spectrometry of Biomacromolecules - Swammerdam Institute of Life Sciences (SILS)
2
Biomolecular Synthesis - Van ‘t Hoff Institute for Molecular Sciences (HIMS)
Universiteit van Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.

Introduction References
(1) Agard, N.J., et al., A Comparative Study of
To answer the question to which extent gene Bioorthogonal Reactions with Azides, ASC
expression is regulated post-transcriptionally, we Chemical Biology 2006, 1, 644-648
have developed a pulse-chase labelling method (2) Kiick, K.L., et al., Incorporation of Azides into
that will allow us to determine the relative rates of Recombinant Proteins for Chemoselective
protein synthesis and degradation. Newly Modification by the Staudinger Ligation, PNAS
synthesized proteins are labelled with a bio- 2002, 99, 19-24
orthogonal methionine analogue, bearing an azide
(see figure 1), and selectively captured on a
specially designed resin (figure 2A). Subsequent
MS analyses will provide identity of proteins and
their de novo translational rates. Here we show the
selective enrichment of azide-containing peptides
from different origin.

Methods
Figure 1, Azidohomoalanine is an analogue of
The designed affinity purification method was methionine and readily accepted by methionyl-
tested for a single (synthetic) peptide containing tRNA synthetase in Escherichia coli and
azidohomoalanine (Azhal) and then applied to incorporated into proteins (2).
digests of an Azhal-labelled single protein and an
A
Azhal-labelled Escherichia coli proteome. Azide-
containing peptides were selectively captured on
the Cyclooctyne-resin (Cyco) (figure 2A) via the
strain-promoted [3+2]-cycloaddition (1). Coupled
peptides were released upon reduction and
alkylation of the disulphide-linker (figure 2B). The B
enriched Azhal-peptide mixtures were then
analysed by MALDI-TOF and LC-Q-TOF MS(MS).

Results

For a single Azhal-peptide the method is found to

be selective and efficient. For a tryptic digest of a
single protein containing six Azhal residues, the
purification method shows an enormous
enrichment in Azhal-containing peptides. Results
will be presented of the selective capturing and Figure 2, A The Cyco-resin, consists of a poly-
release of Azhal-peptides from the digest of an dimethylacrylamide solid support, a cleavable
Azhal-labelled Escherichia coli proteome. disulphide linker and a cyclooctyne as reactive
group. B Modified peptide after capture and
Innovative aspects release from the Cyco-resin.

• The method uses strain-promoted [3+2] –

cycloaddition on a solid support for the
purification of azide-containing peptides
• The method allows us to discriminate between
previously and newly synthesized proteins and
selective analysis of the latter
• The method is generally applicable for the
purification of azide-containing compounds
from complex mixtures
 Processing Samples for High-Throughput Clinical Mass Spec Using Parallel
Electrophoresis
1 2 2
Jeremy L. Norris ; James A. Mobley ; Greg Bowersock ;
1 1
James B. Harkins, III ; Benjamin B. Katz
1 2
Protein Discovery, Inc., Knoxville, TN; Department of Urologic Surgery, University of Alabama-Birmingham,
Birmingham, AL

Introduction total of 511 unique proteins were identified,

The need for high-throughput sample preparation consisting of 409 cationic proteins and 110 anionic
for clinical proteomics represents a bottleneck to proteins. Only 8 proteins were identified that were
the application of proteomics to clinical common to the two fractions (Fig 2).
diagnostics. A number of sample preparation steps
are required to prepare complex samples for Innovative aspects
proteome analysis by mass spectrometry. These • High-throughput, mass spectrometry
samples preparation steps include concentration, compatible sample preparation platform.
desalting, fractionation, and depletion of high • Integrates depletion, concentration,
abundance proteins. This study will present a fractionation and desalting into single
novel sample preparation device that integrates electrophoretic step.
these steps into a single automated platform. • Programmable MWCO allows for enrichment
Advantages for biomarker discovery applications of the low molecular weight proteome.
will be demonstrated.
References
Methods (1) Harkins, J.B.; et al. Parallel Electrophoretic
Unprocessed samples were prepared for MALDI Depletion, Fractionation, Concentration
and ESI analysis using electrophoretic sample and Desalting of 96 Complex Biological
preparation (1). Anionic and cationic proteins were Samples for Mass Spectrometry. Anal.
separated into unique fraction while Chem. 2008 (ASAP).
simultaneously depleting high abundance, high
molecular weight proteins from the samples. The
proteins were captured onto a reversed-phase
monolithic capture medium and read directly using
a mass spectrometer or extracted for offline
analysis. Samples were analyzed directly using
both MALDI MS and top-down LC/MS/MS.
Biomarker discovery was performed with minimal
sample manipulation, and samples were prepared
for top-down LC/MS/MS using the same sample
preparation program,. The proposed workflow for
biomarker discovery and protein identification will Figure 1: Schematic of the electrophoretic sample
be reviewed. preparation device.

Results Cation Capture

Human serum samples were prepared for analysis
Anion Capture
using electrophoretic sample preparation in 1 h
(Fig 1). Reproducibility was assessed by the
addition of standard proteins to 10 different patient
sera and evaluating the coefficient of variation of 409 8 110
peak area for each at their optimum run time. It
was found that the sample preparation adds little
variation to that achieved for the measurement of
ions using MALDI MS (CV <30%). The response
of these standards spiked into human serum was Cation Capture Anion Capture
found to be linear with respect to concentration total unique total unique
across approximately 2 orders of magnitude. Run Time peptides proteins peptides proteins
1C 227 153 78 57
Attomole-level sensitivities were achieved. The 2C 356 265 86 62
removal of highly abundant proteins (e.g. albumin) 1C+ 2 C --- 409 --- 110
was confirmed offline using 1D gel electrophoresis. Unique Proteins Identified in All Conditions 511
Similarly, proteins were eluted off-chip from
identically prepared fractions and analyzed in a Figure 2: Proteins identified using top-down LC/MS/MS
from human serum.
top-down fashion using capillary LC/MS/MS. A
 Reduction of High Abundance Proteins in Serum, Plasma and Non-Blood Samples
with ProteoMiner Beads

A. Paulus(1), T. Wehr (1), N. Liu(1), K. Academia (1), S. Freeby (1), E. Boschetti (2)

1 Bio-Rad Laboratories, Life Science Group, 6000 James Watson Drive, Hercules, CA 94547
2 Bio-Rad Laboratories, c/o CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France

Introduction
Biomarker discovery projects are typically conducted
with serum or plasma samples. The high dynamic range
of 10–12 orders of magnitude surpasses the capabilities
of existing separation and analysis techniques. Serum
and plasma are dominated by 20 or so high-abundance
proteins that make up 95–99% of the protein mass.
Typically, immodepletion methods are used to remove
high abundance proteins.
We developed an alternative to antibody-based methods
with ProteoMiner™, which is based on treatment of
complex protein samples with a library of hexapeptides
bound to chromatographic supports. Each unique
hexapeptide binds to a unique protein recognition site.
In this presentation, we explore the use of ProteoMiner
for high abundance protein reduction in plasma and
serum in biomarker studies, its ability to retain
quantitative information on medium to low abundance
proteins, applications to non-blood samples and work
with an artificial serum to understand mechanisms of
action.
We also show that the quantitative information for
proteins in the ng/ml range is retained after ProteoMiner
treatment using SAA spiked samples. Finally, we are
using an artificial blood to examine the retention
mechanism of ProteoMiner beads for select known
proteins. We find that we can detect tissue leakage
proteins such as retinol binding protein after
ProteoMiner treatment on 1D and 2D gels.
Innovative aspects
o Novel method to dramatically reduce high
abundant proteins in serum and plasma
samples
o Retention of quantitative information for
medium to low abundance proteins to allow
biomarker discovery
o Application of high abundant protein reduction
in biomarker discovery projects, non-blood
samples and artificial serum

Methods
We subjected human plasma and serum, some from
patients with known disease status of cardiac arrests and
diabetes, to ProteoMiner treatment. Subsequent analysis
was done via SELDI, 1D LC-MS-MS and 2D LC-MS-
MS. In addition, we used Western blots to track
quantitative measurement of select medium to low
abundance proteins such as SAA. Protein identification
was done after spot or band excising and trypsin
digestion using a nanospray LC coupled to a Thermo
LTQ iontrap MS. The artificial serum was composed by
mixing of 17 commercial proteins both in equimolar
amounts and in the mass ratios found in serum and
plasma.

Results
Using ProteoMiner reduces the initial protein mass in
serum and plasma by 97%. The remaining 3% protein
mass represents all proteins including the high
abundance proteins, albeit at a lower concentration in
the sample. The procedure is reproducible as
demonstrated via SELDI and 2D analysis. We show that
ProteoMiner treatment increases the 2D spot count from
about 200 for a control to close to 500 after the
treatment. Applied to cardiac arrest and diabetic
samples, the quantitative difference in expression levels
increases roughly 10 fold compared to a control. Data of
excised spots identified via LC-MS-MS will be shown.
First results of using ProteoMiner to non-blood samples
with the presented using plant and cell extract samples.
 The Secretome of H460 non small cell lung cancer cells: comparing workflows

Sander Piersma, Simone Span, Frank Kruyt, Remond Fijneman, and Connie Jiménez
Oncoproteomics lab and medical oncology, Cancer Center Amsterdam, VU medical center, De Boelelaan
1117, 1081 HV Amsterdam, The Netherlands

Introduction experiments, respectively. The H460 secretome

Biomarker discovery in plasma is hampered by the
plasma proteome complexity as well as its
dynamic range. We focus on a sub-proteome
consisting of the proteins secreted by tumor cells;
the secretome. The secretome has a reduced
complexity compared to plasma, is enriched for
cancer-cell specific proteins and moreover,
secreted proteins are likely to be detected in blood.
The reduced complexity allows for coverage of a
higher dynamic range as well as detection of lower
abundance proteins. Here we describe a
comparison of ID-based LC-MS/MS workflows for
secreted proteins.
Methods
Adhering H460 non small-cell lung cancer cells
were grown, and 24 hours prior to harvesting cells
were washed and cultured on serum-free medium.
Medium was collected and concentrated by
ultrafiltration (10 kDa cut-off). Three methods for
secretome analysis were compared
• protein hydrophobic interaction (C2 resin)
followed by tryptic digestion
• 10% SDS-PAGE or 4-12% SDS-PAGE
followed by in-gel digestion
• solution digestion followed by off-line SCX
chromatography.
The final step for all 3 methods was nanoLC-
MS/MS on a Dionex Ultimate 3000 system
connected to a Thermo LTQ-FT. Data was
searched against IPI-human using Sequest (min 2
peptides, <10 ppm MMD) and ID’s were validated
by applying the Peptide prophet (P>95%) and
Protein prophet (P>99%) algorithms using
Scaffold.
was enriched for secreted proteins compared to
H460 total cell lysate (1726 proteins identified in a
10 gel band IGD experiment) as found by Signal P,
Secretome P and GO analysis. As compared to
literature (1) this data set comprises the largest
Secretome to date. The 4-12% SDS-PAGE LC-
MS/MS analysis was superior over the other
workflows.
Innovative aspects
• Optimized workflow for secreted protein
analysis yielding several-fold more ID’s than
reported in literature (1)
• The secretome represents a sub-proteome
enriched for candidate biomarkers
• The optimized workflow has been successfully
applied to the differential analysis of tumor
tissue secretomes in a colon tumor mouse
model
References
(1) Jiménez et al, High-throughput and
targeted in-depth mass spectrometry-
based proteomics for biofluid profiling and
biomarker discovery; Biomarkers in
medicin 1 (4), 541-565 2007
17
12 54
TC2
416
20 20
17
10 33

Results 29 35 35 121
IGD SCX
One of the key points in obtaining a good 876 693
secretome fraction is replacing the medium by
serum-free medium, thus minimizing interference 37 16 24 47
89 26
by bovine serum-derived proteins. However,
serum-free incubation time has to be optimized to
minimize apoptosis and cell lysis. For H460
NSCLC cells we found 24 hours to be optimal with SCX 45 934 158 IGD
respect to protein yield and cell viability.
We compared 3 workflows. Intact proteins
separation by C2 hydrophobic interaction resin
method yielded 580 proteins (N=3 experiments) Figure 1. Venn diagrams of N=3 biological replicates of
with 416 proteins in all 3 experiments. Conversely, H460 secretomes analyzed by TC2, SCX and 4-12%
the 10% SDS PAGE IGD analysis yielded 895 SDS-PAGE followed by LC-MS/MS. Reproducibility of
proteins (N=3) with 699 proteins identified in all 3 identification was 72% for TC2, 71% for the SCX
experiments. Comparing this with 4-12% gradient workflow and 80% for the 4-12% IGD workflow. The
gel and off-line SCX gave 1092 and 979 identified lower panel shows the overlap between the 2 most
proteins with 876 and 693 proteins in all 3 successful data sets (all protein ID’s in N=3 included)
 ALTERNATIVE TWO DIMENSIONAL ELECTROPHORESIS – OFFGEL
ELECTROPHORESIS COMBINED WITH HIGH SENSITIVITY MICROFLUIDIC ON-CHIP
PROTEIN DETECTION

Tobias Preckel, Andreas Ruefer, Christian Wenz, Martin Greiner, Agilent Technologies R&D and Mktg.
GmbH & Co.KG, Hewlett Packard Strasse 6, Waldbronn, Germany

Introduction the fraction of time required for conventional 2D-

GE.
Two dimensional gel electrophoresis (2D-GE)
employs isoelectric focusing in the first dimension
and a separation of the proteins according to their Innovative aspects
molecular weight in the second dimension. The
gels are then stained using silver stain to visualize • Reproducible, easy to use, standardized
the protein pattern. This method is unrivalled in methodology
terms of resolution but is a tedious and time- • Detection of expression changes of 1%
consuming procedure. Here we present a possible
combination of two easy methods that separate
proteins in analogy to 2D-GE according to their
isoelectric point (pI) and molecular weight (kDa). References

1. Michel P. E., Reymond F., Arnaud I. L.,

Methods Josserand J., Girault H. H. and Rossier J. S.,
“Protein fractionation in a multicompartment device
using Off-Gel isoelectric focusing.” Electrophoresis
For the first dimension, OFFGEL electrophoresis
24, 3-11, 2003.
was used. This newly developed method takes
advantage of the impressive resolving power of
2. Hörth P., Miller C. A., Preckel T. and Wenz C.,
immobilized pH gradient gel based isoelectric
“Efficient fractionation and improved protein
focusing (IPG IEF) but in contrast to conventional
identification by peptide OFFGEL electrophoresis.”
isoelectric focusing delivers sample in liquid phase
Mol. Cell. Proteomics 5, 1968-1974, 2006.
thus avoiding sample recovery from the gel. For
the second dimension, a microfluidic high
sensitivity on-chip protein sizing method was
employed. This method allows separating proteins
from 5 to 250 kDa and offers a sensitivity
equivalent or better than silver staining and a
linear dynamic range across four orders of
magnitude.

Complex protein samples, e.g. E.coli protein

lysate, were first fractionated by OFFGEL
electrophoresis by pI in the pH range 3-10.
Individual fractions were then separated by size
and analyzed by microfluidic on-chip protein
detection with high sensitivity.

Results

The combination of the two technologies is

possible to detect a small change in protein
pattern, comparable to the absence and presence
of only 1% protein relative to the total protein
concentration. Combining the resolution power of
OFFGEL electrophoresis in the first dimension with
the high sensitivity on-chip electrophoresis in the
second dimension it is possible to obtain a pseudo
2D gel image in a more automated manner and in
Fast and versatile : fluroescent staining of protein in gels with oxacarbocyanines
Mireille Chevallet 1 , Hélène Diemer 2, Alain Van Dorsselaer 2, Thierry Rabilloud 1
1 : Biophysique et Biochimie des Systèmes Intégrés, iRTSV/ LBBSI, CNRS UMR 5092, CEA Grenoble,
17, rue des martyrs, 38054 Grenoble France
2 : Laboratoire de Spectrométrie de Masse, UMR CNRS 7178, Strasbourg
Introduction :
The technical challenges associated with post-
electrophoretic detection of proteins in SDS gels
(and in 2D gels) are numerous. They encompass
sensitivity, linearity, homogeneity and now
compatibility with downstream procedures. When
large series of gels are to be run, cost-efficiency
and safety can also become issues.
We describe here a new, versatile fluorescent
detection method, using commercial
oxacarbocyanines as SDS-sensitive
fluorophores. Depending on the user’s need, this
method can be implemented either as a fast,
non-fixing metod or as a long, stable,post-
fixation method
Methods :
Reagents
dialkyloxacarbocyanines of various chain lengths
(C2-C18) were dissolved in DMSO at 30 mM.
Electrophoresis
For 2D gels, IEF was carried out on IPG strips.
The SDS dimension used either the Laemmli
system (ionic strength 62.5 mM) or the taurine
system (1) (ionic strength 0.1M)
Detection
For the fast, non-fixing staining, the
carbocyanine was mixed in the cathode buffer, at
a concentration of 3 μM. After electrophoresis,
the gel was washed in water for 15 minutes
(laemmli system) or 3x15 minutes (taurine
system).
For the post-fixation staining, the gels were fixed,
washed in 2% phosphoric acid, 0.004% SDS,
and then stained for 4 hours in the same solution
containing 3 μM of the desired carbocyanine.
Proteins were visualized on a 302 nm UV table
or with a laser scanner (488 nm excitation)
Results
When compared to classical detection methods
(colloidal blue, silver staining, Sypro Ruby) the
carbocaynine detection method shows the
following features :
-Non fixing when the fast, co-electrophoretic
setup is chosen. Proteins can be moved out of
the gel (e.g. for blotting)
-Sensitivity, linearity and homogeneity close to
Sypro Ruby, in both formats (fixing or non-fixing)
-Low cost
-Excellent compatibility with mass spectrometry
The optimal carbocyanine depends on the
staining method chosen. Hexyl or heptyl
carbocyanines are optimal for co-electrophoretic
staining, while ethylcarbocyanine is the best
choice for post-fixing staining.
Depending on the conception of the
experiments, fixing or non-fixing format can be
selected. The stain is stable (but proteins are
insolubilized in the gel) in the fixing format. In the
non-fixing format, the stain is stable for a few
hours, but the proteins are not precipitated. This
format is therefore useful prior to protein blotting,
for GeLC experiments, or when a fast answer is
required.
As the mechanism of staining is through
fluorescence enhancement in lipophilic
environment (here the SDS-protein complexes),
no protein modifications are seen
Innovative aspects
Good combination of sensitivity, cost-efficiency,
and compatibilty with downstream analytical
procedures
Versatility through the choice of fixing or non
fixing methods
References
(1) C. Tastet et al. A versatile electrophoresis
system for the analysis of high- and low-
molecular-weight proteins. Electrophoresis 2003
24, 1787-1794
(2) S. Luche et al. Ultrafast coelectrophoretic
fluorescent staining of proteins with
carbocyanines Proteomics 2007 7, 3234-3244
 Highly efficient depletion strategy improves erythrocyte proteome coverage
dramatically

Jeffrey H. Ringrose, Wouter W. van Solinge, Shabaz Mohammed, Martina C. O’Flaherty, Albert J.R. Heck,
and Monique Slijper
Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, Sorbonnelaan 16, 3584 CA
Utrecht, The Netherlands, Fax (+31) 30 2518219, e-mail: j.h.ringrose@uu.nl

Introduction detect 700 unique proteins, which is a

Hemoglobin (Hb) makes up for 97% of the total dry considerable increase compared to the 167
mass of RBCs. This makes identification of low detected proteins before depletion. In conclusion,
expression level proteins extremely difficult (1). We using a double-depletion approach the number of
developed a very efficient Hb-affinity column to identified proteins in soluble RBC lysate increased
deplete for Hb. We combined this with ion dramatically. Our strategy is useful in any
exchange (IEX) chromatography to deplete for the proteome study hampered by the presence of
second most abundant protein which is carbonic hemoglobin or CA-1. It also opens up new
anhydrase 1 (CA-1). Using this strategy, we perspectives to uncover erythrocyte disorders with
drastically improved the proteome coverage of the unknown etiology.
soluble erythrocyte proteome. This opens up new
perspectives for the study of the RBC proteome of Innovative aspects
hemolytic anemias with unknown causes, and the • Highly efficient and specific depletion of
development of diagnostic tools. haemoglobin and carbonic anhydrase-1 from
erythrocytes or any sample contaminated with
blood.
Methods
EDTA-blood was passed over an alpha-cellulose References
column. Washed RBCs were lysed by hypotonic (1) Goodman et al. The human red blood cell
shock and cleared by centrifugation. RBC soluble proteome and interactome. Exp Biol Med
protein was passed over a sepharose based Hb 232:1391-1408,2007)
depletion column. IEX was performed using in
series connected Strong Cation and Anion
Exchange columns using step elution with NaCl.
Tryptic digests from SDS-PAGE gel pieces were
analyzed by LC-MS/MS using a LTQ or hybrid
LTQ-FT-ICR mass spectrometer. MS/MS data
were analyzed with Mascot search engine version
2.2.0 (Matrix Science, London, UK) and the IPI-
Human-3.28 database. Protein probabilities were
accepted at 99.9% as determined by Protein
Prophet using Scaffold software (Proteome-
Software, Portland, USA). The false discovery rate
was calculated to be around 0.5% using the
automatic decoy function of Mascot.

Results
We developed an efficient and specific Hb
depletion column based on sepharose. SDS-
PAGE analysis revealed that after Hb depletion the
number of visible protein bands was greatly
increased (Figure 1). Using IEX chromatography
we could also deplete for CA-1 which after Hb
depletion became the most abundant protein.
Under the conditions used the CA-1 was present in
Figure 1. SDS-PAGE analysis of RBC lysate (lane1), Hb
the IEX flow through whereas almost all other affinity column bound fraction (lane 2), Hb depleted RBC
proteins were retained and subsequently eluted fraction (lane 3), CA-1 containing IEX flow-through
from the columns. We analyzed all fractions by fraction (lane 4), and double depleted RBC fraction (lane
SDS-PAGE and subsequent MS/MS. The number 5). Coomassie stained gel.
of identified proteins from RBC lysate increased
from 167 before depletion to 677 after depletion for
both Hb and CA-1. In the Hb and CA-1 fractions
we detected 20 and 39 proteins respectively.
Using our depletion strategy we could in total
 Guided Gas-Phase Fractionation: a Strategy to Tackle Duty-Cycle Limitations

Anne Rokka (1), Aschwin van der Woude (1), Santosh Gupta (2), Matthias Nees (2), and Garry Corthals (1)
1)Turku Centre for Biotechnology, University of Turku & Åbo Akademi University,
Tykistökatu 6A, 20520 Turku, Finland
2) Medical Biotechnology, VTT Technical Research Centre of Finland and
University of Turku, Turku, Finland

Introduction
The relationship between the duty-cycle of an MS Innovative aspects
instrument and the number of peptides that can be • Easy peptide pre-fractionation by IEF
identified from a single LC-MS experiment is • Improved protein identification by guided
relatively straightforward: the faster the duty-cycle, GFP
the more peptides will be identified for a given • Quality assessment on any LC-MS run by
data-dependent acquisition experiment. For most pepRecon tool
proteome-scale experiments we are severely
limited in our analyses, and attempts at complete
analysis are not immediately foreseeable. Thus we
face a formidable challenge in developing methods
that make the step towards full-scale analysis.

Methods
In an attempt to maximize proteome coverage we
used an integrated approach that allowed for
gentle fractionation of peptides in to two phases:
the liquid-phase and gas-phase. Liquid-phase
fractionation was achieved by peptide isoelectric
focusing (pepIEF) prior to MS analysis and gas-
phase fractionation (GPF) was achieved in the MS
instrument.

Results
Peptide isoelectric focusing (pepIEF) was used for
pre-fractionation of the complex peptide sample
prior to MS analysis because it is simple,
reproducible and do not involve chemical or
structural modifications to peptides and is suitable
for practically any proteomics approach. Its
performance as a pre-fractionation step for
peptides prior to LC-MS-MS is less common, even
though it is as successful as commonly employed
SCX procedures. Three of the twelve IEF fractions,
containing either acidic, neutral or basic peptides,
were chosen for GPF analyses.
We found GPF as a simple analytical approach
that can be used to increase the number of
identifications without the need for hardware
modifications. GPF iterative LC-MS/MS analyses
of a sample were performed on selected m/z
ranges that had been determined following a
normal wide m/z range scan (typically 400–1800
m/z). In other words, the proteome complexity was
determined empirically prior to GPF. We have
written a suite or R scripts that enable the detailed
and rapid evaluation of empirical data and
concurrent quality assessment of any LC-MS run.
We will present data on the principle and
application of guided GPF and give examples of
how a massive increase in the number of
identifications is achieved for peptide
identifications.
 Proteomics on a chip for monitoring autoimmune diseases

Richard B.M. Schasfoort, Bianca Beusink, Dietrich Kohlheyer, Angelique Lokate, Ger Pruijn, Ad de Jong,
Wout van Bennekom, Linda Silvertand, Natasja Carol-Visser, Albert J.R. Heck and Albert van den Berg.
BIOS Lab on a chip Group, MESA+ Institute for nanotechnology,
P.O. Box 217, 7500 AE Enschede, the Netherlands

Introduction can be carried out using the FFE-SPR technology.

The research in the “Proteomics on a chip for The state of the art will be shown at HUPO 2008.
monitoring autoimmune diseases” project carried
out at the University of Twente, University of Innovative aspects
Utrecht and the Radboud University of Nijmegen is 1. New instrument for measuring label free
driven by the need for alternative technologies for diluted patient sera using Surface Plasmon
separation and analysis of the highly complex Resonance imaging. Contacless spotting of
protein mixtures that are inherent to proteomics SPR sensor surfaces monitoring autoimmune
research. The primary objective is to develop an responses of diluted patient sera.
integrated “proteomics on a chip” device for 2. Proteomics on a chip based on FFE-SPR for
multiparameter biomarker screening of functional characterisation of specific
autoimmune patients. The second goal of the interactions and epitope mapping.
project is to discover new autoimmune targets and 3. Characterization and identification of
applications based on these autoantigens. We fibrinogen digest mixtures in a combination
developed a new combination of a separation with isoelectric focussing (IEF) FFE-SPR and
technology based on Free Flow Electrophoresis ESI-MS
(FFE) and iso-electric focussing (IEF) in
combination with Surface Plasmon Resonance References
(SPR) imaging. 1. Lokate, A.M.C. et al. (2007) JACS, 129 (45) 14013
2. Beusink J.B. et al. (2008) Bios&Bioel. 23 (6) 839
3. Kohlheyer D. et al. (2007) Anal. Chem. 79 (21) 8190
Methods
For the primary goal of the project a 24-spot
microarray was developed containing human IgG
as well as two different linear citrullinated peptides
(CitA and CitB) and the corresponding control
peptides (ArgA and ArgB, containing arginine
instead of citrulline), (1 nanoliter per spot) on a N-
hydroxysuccinimide pre-activated polycarbo-
xylate-coated gold sensor surface using a non-
contact spotting instrument of Biofluidix, Freiburg
(D) (Top Spot). A diluted patient serum (100*) was
exposed to the microarray and the biomolecular
interactions were measured using the scanning
imaging SPR instrument (IBIS-iSPR of IBIS
Technologies) [1]. For the second goal we
integrated a FFE device with SPR imaging. Figure 1 . Sensorgram (raw data) showing biomolecular
Autoimmune responses to citrullinated fibrinogen interactions at several ligand and analyte concentrations
and its peptides were measured and MS for determination of the affinity and rate constants using
identification was carried out. an automated liquid handling procedure from [2].

Results buffer medium

electrode sample
The reactivity of sera was quantified by calculating
the ratio between the angle shifts observed for the V+
citrullinated peptide A and the corresponding buffer medium
arginine control peptide binding of serum
antibodies. The results for 50 RA sera and 29
control sera were measured successfully [1]. In V-
figure 1 the results of sequential interactions of
serial diluted analyte to ligands spotted in several
SPR sensor region
densities are shown in a single sensorgram. flow direction
Autoimmune responses of citrullinated fibrinogen
were measured using the FFE-SPR approach. A
typical device is shown in figure 2. It is foreseen
Figure 2. Free Flow Electrophoresis chip including
that epitope mapping based on fibrionogen digests
Surface Plasmon Resonance detection
Use of a novel hydrophilic-PVDF membrane for the immunodetection of
phosphorylated proteins separated by gel electrophoresis

Protein phosphorylation is the major mechanism by which diverse cellular processes are
regulated. Despite its importance, the paucity of phosphorylated proteins in a biological
sample poses a major hurdle in studying these processes. Immunodetection method,
based on the use of phospho amino acid specific antibodies to detect phospho-proteins
blotted onto either polyvinylidene fluoride (PVDF) or nitrocellulose (NC) membranes,
offers highly sensitive yet relatively simple way of studying global protein
phosphorylation.

We describe here a new water-wettable, low auto-fluorescent, PVDF membrane and

show its use for the study of protein phosphorylation.

A431 cell lysates (EGF stimulated and non-stimulated) were separated by SDS-PAGE
and blotted onto either hydrophilic PVDF or NC membranes. Analysis of global p-Tyr/p-
Ser proteomics were performed by western immunodetection using monoclonal antibody
4G10 (p-Tyr) and recently developed p-Ser monoclonal antibody 4A4. Two different
detection methods were employed: traditional chemiluminescence or multiplexed
fluorescent detection using a near infrared laser scanner.

Results demonstrate hydrophilic PVDF membranes exhibit instant and uniform wetting in
water and aqueous buffers without the alcohol pre-wetting step, while maintaining a level
of immunodetection performance comparable to NC membranes. In contrast to the
conventional PVDF membrane, the new membrane exhibited substantially reduced
background auto fluorescence making it suitable for multiplexed fluorescent
immunodetection. Use of hydrophilic PVDF membrane offered selective and sensitive
detection of serine and tyrosine phosphorylation when combined with 4A4/4G10
immunodetection, enabling global profiling of protein phosphorylation. While shown its
utility in EGF signaling pathway in A431 cells, the combined use of hydrophilic PVDF
membrane with phospho-immunodetection can be easily applied to other biological
systems.
 Observing Rapid H/D Exchange of The Reactive Hydrogen on the Protein Molecules
by Electrospray-Assisted Laser Desorption Ionization (ELDI) Mass Spectrometry
1
Jingyueh Jeng
2,3 2 2 2
Jentaie Shiea , Li-Hua Lo , Yi-Tzu Cho , Cheng-Hui Yuan
1
: Department of Biotechnology, Chia Nan University of Pharmacy & Science
2
: Department of Chemistry, National Sun Yat-Sen Univ., Kaohsiung, TAIWAN
3
: National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, TAIWAN

Introduction slight differences in sequence and structure of the

Electrospray-assisted laser desorption ionization two protein standards. The H/D exchange number
(ELDI) mass spectrometry is a useful technique to of peptides and proteins decreased with the
characterize proteins under ambient conditions. In decrease of the ratio of D2O/H2O for ESI. A
ELDI, the solid sample was irradiated with a laser reverse H/D exchange experiment was also
beam and the desorbed molecules were ionized in performed by using H-solution for ESI and dried
an ESI plume to give an ESI-like mass spectrum. deuterated proteins as the samples.
Since desorption and ionization in ELDI is
separated events, the composition of the ESI Innovative aspects
solutions can be modified for particular The number of reactive hydrogen in the protein
applications. In this study, the deuterated solution molecule was determined by rapid H/D exchange
was used to generate electrospray. It was using ELDI/MS.
demonstrated that, once the laser-desorbed
protein molecules entered into the deuterated ESI
plume, not only ionization but H/D exchange of the
desorbed protein would occur. The cytochrome c
with different origin, sequence, and structure could
were rapidly distinguished.

Methods
Aqueous sample solutions containing protein
standards were prepared. Ten microliters of the
sample solution were deposited on a stainless
steel sample plate by a micropipette. After being
dried, the sample spot was irradiated with a pulsed
nitrogen laser beam. The laser- desorbed analyte
molecules entered an ESI plume generated from a
capillary located 5 mm above the sample spot. The
solution containing different ratio of D-solvents
(D2O, MeOD, and CH3COOD) and H-solvents
(H2O, MeOH, CH3COOH) was used for
electrospray. The protein ions were detected by an
ion trap mass analyzer attached to the ELDI
source.

Results
Hypothetically, the time between molecular
desorption and ion production in ELDI is estimated
to be in milliseconds. This time scale will only allow
the reactive or outmost hydrogen atoms on the
desorbed protein molecule to be exchanged with
the deuterium in the ESI plume. Hydrogen atoms
in peptide bonds and secondary amines showed
no H/D exchange, for they required longer time to
exchange with the deuterium. It was found that the
H/D exchange number of cytochrome c in the ELDI
was much less than that found through
conventional methods. However, the exchange
H/D number of the cytochrome c with different
origin shows slight differences. For example, 42
hydrogen atoms were exchanged for the
cytochrome c from a rabbit and 46 atoms for the
cytochrome c from a horse. This may be due to
 Top-Down Proteomics analysis using Automated 2D LC and Fractionation
Evert-Jan Sneekes, Bas Dolman, and Remco Swart
Dionex Corporation, Amsterdam, The Netherlands

Introduction Innovative aspects

The complex nature of proteomic samples requires • Application of Polymeric monolithic columns
high resolution separation methods prior to MS for intact protein separations
detection. Multidimensional LC separations of • Automatic off-line 2D-LC followed by automatic
proteins have been developed, however gel in-well digestion to speed up gel free top-down
electrophoresis remains the dominant separation proteomics analysis.
technique for intact proteins, mainly for the
resolution and visualization it provides. Liquid
phase top-down workflows would offer easier
sample transfer to subsequent analysis, but so far
could not deliver an appropriate alternative to the
advantages of gel based approaches.
Here we present a fast, fully automated top-down
LC-based workflow.

Methods
We describe an automated two-dimensional LC
workflow for the separation and identification of
proteins. The workflow involves a first dimension
ion-exchange separation and second dimension
reversed-phase separation, both performed on
polymeric monolithic columns and both dimensions
were fractionated in well plates. The application of
monolithic columns allows for fast and highly
efficient protein separations and the resulting
chromatograms are presented in a 2D retention
map. In-well digests of selected fractions are
subsequently analyzed by capillary LC-MS/MS for
their identification. This LC based top-down
workflow was validated with an E.coli protein
extract and the recently introduced Universal
Proteomics Standard Set (Sigma).

Results
The complete 2D LC separation of intact proteins
was achieved in 4 hours, including fractionation of
both first and second dimension. Tryptic digestion
was performed immediately after the second
dimension fractionation and quenched after 3
hours. A 2D retention map was generated based
on the UV data of the protein separations and was
used to select fractions of interest.
The tryptic peptides in these fractions were then
analyzed by capillary LC-MS/MS for protein
identification. Evaluating the analyzed fractions
revealed a high efficient protein separation with
minimal overlap between fractions. In well
digestion of the collected LC fractions and
subsequent LC-MS/MS analysis provided
confident protein identifications.
 Optimization of separation power in one-dimensional LC
in analysis of proteomics samples

Bas Dolman, Sebastiaan Eeltink, Remco Swart

Dionex Benelux, Abberdaan 114, 1046 AA Amsterdam, the Netherlands

Introduction
9.00
One of the main challenges in proteomics research mAU WVL:214 nm

with nanoLC-MS is to improve the quality of the

separation of highly complex samples prior to MS
detection. Especially the use of longer gradients 6.00

and longer columns are attractive ways to improve

the separation. 4.00

In this communication we describe the optimization

of LC conditions and column technology to obtain 2.00

the maximum separation power in one-

min
dimensional LC. 0.00
25 40 60 80 100 125

Figure 1. Separations of a tryptic digest of 6 proteins

Methods
performed at optimized LC conditions on a 75 μm x 25
The nano-LC performance is characterized by the cm column packed with 3 μm silica C18 particles.
maximum number of peptides that can be
separated with a resolution of 1 within a certain Innovative aspects
gradient time (peak capacity). Peak capacity was
• Maximizing peak capacity of peptide
maximized by optimizing the LC conditions, such
separations by optimizing column technology.
as gradient time, flow rate, and temperature and
the column technology. The impact of these • Demonstration of LC performance in terms of
parameters was experimentally evaluated in detail peak capacity versus analysis time.
for the separation of complex peptide mixtures in
nano-LC mode.

Results
Using 15 cm long columns packed with 3 μm silica
C18 particles the effect of gradient time on peak
capacity and peak width was investigated. Longer
gradient times yielded higher peak capacities. A
steep initial increase in peak capacity is observed
when increasing the gradient time from 5 to 40
min. At longer gradient times the rate of increase is
less steep, and peak capacity tends to reach a
maximum around 300, which is caused by the
increase in average peak width of peptides at
longer gradient times.
With increasing temperature from 25 to 40°C a
strong increase in peak capacity was observed
(∼35%). At elevated temperatures the viscosity is
reduced and diffusion in the mobile and stationary
phase is greatly enhanced, leading to narrower
peaks.
In addition, the effect of column length on peak
capacity was investigated. In general, longer
columns yielded higher peak capacities. An
example of the separation power of long packed
columns at optimized LC conditions in one-
dimensional LC is shown in Figure 1. Over 320
different peptides were separated within 125 min.
 Study of β-Catenin-associated Complex by Nanoprobe-Based Affinity Mass
Spectrometry

1 1,2 3 3 4 1
An-Kai Su , Hsin-Hung Huang , Po-Chiao Lin , Chun-Cheng Lin , Jeou-Yuan Chen , and Yu-Ju Chen
1,4 2
Institute of Chemistry and Biomedical Sciences, Academia Sinica, Institute of Biochemical Sciences,
3
National Taiwan University, Department of Chemistry, National Tsing Hua University, Taiwan

Introduction
As well known, β-catenin involves in the Wnt References
pathway and acts as a transcriptional co-activator (1) Lin et al, Ethylene glycol-protected
in the control of cell proliferation and migration magnetic nanoparticles for a multiplexed
during metastasis. Cancer metastasis is regulated immunoassay in human plasma; Small, 2,
by complex interplay between the β-catenin- 485-489, 2006.
mediated adhesion pathway and β-catenin/TCF
complex formation within the nucleus. Taking
advantages of high surface-to-volume ratio and
easy separation of magnetic nanoparticles (MNPs),
we present a nanoprobe-based affinity mass A B
pre-clean + + + +
spectrometry (MS) strategy that simultaneously _ _ _
pre-clean + MNPs + +
isolate and identify β-catenin-associated protein Ab@MNPs + + Ab@MNPs _ + _ +
complexes in gastric cancer cell.
kDa
Methods 250
Antibody of β-catenin was conjugated on MNPs
surface and the MNPs were incubated with lysate
130
of the tumorigenic gastric cancer cell AZ521 for
extraction of β-catenin-included protein complexes. β-catenin
95
The synthesis of antibody-conjugated magnetic
nanoparticles (Ab@MNPs) can be found in the 72
literature (Lin et al. Small, 2, 485-489, 2006). After
incubation, the extracted multiplex protein
complexes were directly analyzed by SDS-PAGE 55
separation for trypsin-digestion and identified by
LC/MS/MS, thereby, achieving multiple protein
screening and the characterization of complex
variants. Figure 1. Enrichment of β-catenin and its associated
protein complex with Ab@MNPs in AZ521 cells. (A) The
silver staining result showed that nonspecific binding
Results proteins were pre-cleaned with MNPs only. (B)
As shown in SDS-PAGE analysis in Figure 1, the Compared with MNPs, amounts of β-catenin was
preliminary results show that using Ab@MNPs can enriched using Ab@MNPs precipitation after MNPs pre-
enrich β-catenin and its interacting partners (Fig. cleaning by western blotting (left). Patterns of
1B). The binding capacity of Ab@MNPs and IP precipitated proteins were compared by silver staining
beads has been compared; the former strategy (right). Western blot analysis was performed using anti-
demonstrated easier purification process for the β-catenin IgG.
follow-up MS identification. In addition, nonspecific
binding of MNPs (Fig. 1A) cab be improved by pre-
clean step with blank MNPs and washing steps
(Fig. 1A). The new analytical method will be
applied to study the different β-catenin-associated
complexes in cancer cells with different metastasis
potential. We expect that the newly developed
assay may have implementations to study
bimolecular interactions in targeted proteomics.

Innovative aspects
• Develop a nanoprobe-based proteomic assay
for simultaneously enrich and identify protein
complexes associated with cancer metastasis.
 A Facile Buffer Exchange Interface
for On-line Hyphenation with Trypsin Microreactor
1,2 1,2 1,2 1 1 1
Liangliang Sun , Xiaoqiang Qiao , Dingyin Tao , Zhen Liang , Weibing Zhang , Lihua Zhang *,
1
and Yukui Zhang
1
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics,
Chinese Academy of Science, 457 Zhongshan Road, Dalian 116023, China
2
Graduate School of Chinese Academy of Sciences, Beijing 100039, China

Introduction
For shotgun method, protein digestion is one of the
most important steps, which is usually performed
in solution, but recently more focus is put on the
trypsin microreactor because of the high digestion
efficiency. However, in most cases, the pH and
organic reagent concentration of protein solutions,
especially for eluants from HPLC separation, are
not compatible with immobilized trypsin. Therefore,
it is indispensable to develop on-line buffer
exchange interfaces (1). In our work, a novel on-
line buffer exchange interface based on
microdialysis was developed, and successfully
applied into the adjustment of pH and acetonitrile
concentration successfully within 30 seconds.
Innovative aspects
• A facile microdialysis interface for adjusting pH
and acetonitrile concentration of protein
solutions within 30 seconds.
• On-line hyphenation of the buffer exchange
interface with trypsin Microreactor.
• Enrichment of proteins during buffer exchange
process.
References
(1) David C. Schriemer et al, Blending protein
separation and peptide analysis through real-
time proteolytic digestion; Anal. Chem. 2005,
77,1572-1579.

Methods
The microdialysis interface for on-line buffer 50
exchange was made of hollow fiber membrane Cytochrome c
with MWCO 3000, and the dead volume was about
2 μL. The counterflow exchange buffer was 40
applied to accelerate the mass transfer and
improve the buffer exchange performance. In our
30
experiments, 0.1% trifluoroacetic acid and
Signal

acetonitrile were used to evaluate the buffer B

exchange performance, respectively. Cytochrome 20
c dissolved in 95% acetonitrile and 0.1%
trifluoroacetic acid was used to investigate the A
protein digestion after buffer exchange. The 10 C
samples after digestion were analyzed by HPLC.
0
Results
Our experimental results demonstrated that with
0 10 20 30 40 50
such a microdialysis interface, buffer with pH 3
Time (min)
could be adjusted to pH 8.0, and 100% acetonitrile
could be decreased to 10% within 30 seconds.
The adjusting results were stable and repeatable, Figure 1. Analysis of blank sample (A, 0.05 mg/mL
which could meet the requirements of protein cytochrome c), digested products by trypsin in solution
(B, 0.1mg/mL cytochrome c), and by trypsin
digestion.
microreactor after buffer exchange (C, 0.01 mg/mL
After on-line buffer exchange, cytochrome c (0.01 cytochrome c).
mg/mL) dissolved in 95% acetonitrile and 0.1%
trifluoroacetic acid, as well as cytochrome c (0.1
mg/mL) dissolved in 100 mM NH4HCO3, could be
digested completely by trypsin microreactor, as
shown in Figure 1. Furthermore, by comparing the
peak height of peptides, it could be seen that
proteins were also enriched during the buffer
exchange. All these results demonstrate that such
a microdialysis interface is of promising in on-line
employment of trypsin microreactor for high
throughput analysis of proteomes.
Protein fractioned by Microscale Solution Isoelectric Focusing and Identification by
μHPLC-MS/MS
Dingyin Tao, Jicheng Duan, Zhen Liang, Weibing Zhang, Lihua Zhang and Yukui Zhang
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese
Academy of Sciences, Dalian, China

Introduction μRPLC, and protein identification by MS/MS, more

Comprehensive analysis of whole proteomes is an protein information could be obtained for proteome
extraordinary challenge because of the complexity study.
and wide dynamic range of proteins. The success
of proteome analysis is highly dependent on the Innovative aspects
quality of sample prefractionation prior to the • Effective method for protein prefractionation by
identification by MS/MS. In our recent work, PMSIEF with high protein recovery and less
preparative microscale solution isoelectric focusing protein overlapping
(PMSIEF) was used to achieve narrow-band • A simple way to improve peptide separation by
fractionation of proteins from Escherichia coli (E. coupled several short columns to a long one to
coli). Proteins in various pH related fractions were perform μRPLC -MS/MS.
further separated using coupled long reversed • A promising technique to combine PMSIEF
phase high-performance liquid chromatography with μRPLC -MS/MS for proteome study
(RP-HPLC) columns and identified by MS/MS.
References
Methods (1) Niroshini N., et.al., J. Proteome Res. 2007,
1.95 μg of E.coli whole cell lysates were separated 6, 3780-3787;
into five fractions by a commercial PMSIEF device (2) Masahito M., et.al., J. Proteome Res. 2007,
from Invitrogen (Carosbad, CA, USA) within 4 6, 6380-3690;
hours (1, 2). The proteins of each fraction were
firstly precipitated by acetone, and then
redissolved in 50 mM Tris-HCl buffer (pH 8.0).
Finally, trypsin was added with weight ratio of
trypsin to protein at 1/25 and incubated at 37 ºC for
40 hours. Three different length columns (5 cm, 10
cm and 15 cm of 300 μm I.D.) were combined
together through two-port unions to form a long
column of 30 cm. The digestion product of each
fraction was further separated by μRPLC prior to
Duo
the protein identification by a LCQ detector.

Results
In PMSIEF, 5 chambers with well-defined pH
range, pH 3.0-4.6, 4.6-5.4, 5.4-6.2, 6.2-7.0 and
7.0-10.0, were generated by isoelectric
membranes, which could be used for protein
fractionation according to their isoelectric points Figure 1. Number of identified unique peptides and
(pIs). Proteins focused in each chamber were proteins in each fraction and in total
digested, and further separated and identified by
long column μRPLC-MS/MS. The coupled long
column could obviously improve the separation
efficiency of peptides. Even with single
dimensional separation by μRPLC, 202, 263, 282,
203 and 181 proteins were respectively identified
in each chamber (as shown in Fig. 1),
corresponding to 835 unique proteins identified
from E. Coli. The detailed information was shown
in Fig. 1. The overlapping of proteins in neighbor
chambers was shown as Fig. 2, and the average
percentage of unique proteins identified by each
fraction was calculated to be 73.82%, In addition,
compared to gel based protein separation, the
protein recovery as high as 95.06% was obtained,
which was quite useful for the analysis of low Figure 2 Protein overlapping of identified proteins in
abudnace proteins. In conclusion, with the each fraction chamber.
combination of off-gel protein prefactionation by
PMSIEF, peptide separation by long-column
 An Alternative 2D-LC-Separation Scheme for Shotgun Proteomics Coupled to
ESI or MALDI MS/MS

A. Tholey, Technische Biochemie, Saarland University, 66123 Saarbrücken, Germany

1 2 2 1 1 2,3
Maria Lasaosa , Nathanael Delmotte , Katja Melchior , Thomas Jakoby , Elmar Heinzle , Christian Huber
1 2
Technische Biochemie, Instrumentelle Analytik & Bioanalytik, Saarland University, 66123 Saarbrücken,
3
Germany, Salzburg University, Austria

Introduction Comparison of platform (ii) and (iii): The

A commonly applied multidimensional separation
strategy for shotgun proteome analysis, in which
the complete proteome is digested and
subsequent separation is conducted at the peptide
level, involves the combination of strong cation
exchange (SCX) and ion pair-reversed phase (IP-
RP) HPLC. Here we introduce and evaluate an
alternative 2D-separation scheme encompassing
st
separation of peptide mixtures by RP-HPLC in 1 -
nd
and IP-RP HPLC in 2 -dimension. The novel
separation scheme was coupled either online to
ESI ion trap or offline to MALDI TOF/TOF MS, the
results of both analytical platforms were compared.
Methods
The cytosolic proteome of Corynebacterium
glutamicum, a bacterium used both for the
biotechnological production of amino acids as well
as a model organism for pathogenic bacteria, was
investigated. After proteome isolation and tryptic
digestion, the sample was split and analyzed using
three different analytical platforms: (i) separation
nd
by SCX x IP-RP HPLC, online coupling 2 -
dimension to ESI ion trap MS; (ii) separation by RP
nd
x IP-RP HPLC, online coupling 2 -dimension to
st
ESI ion trap MS; elution in 1 -dimension was
performed using a water/acetonitrile (ACN)
gradient at pH 10, no ion pairing reagent was
added; (iii) separation scheme as in (ii) but
coupled offline to MALDI TOF/TOF MS. The
nd
conditions for separation in 2 -dimension (IP-RP,
0.1% TFA/ACN-gradient at pH 2, monolithic
polystyrene-divinylbenzene column) were identical
in all three platforms.
Results
Comparison of platform (i) and (ii): The separation
st
of peptides by SCX in 1 dimension lead to the
well-known charge clustering, which caused the
need for adaption of fraction lengths on the
sample. In contrast, separation on a C18 column
and elution with a water/ACN-gradient at pH 10
(pH triggered by Et3N/acetic acid) led to a uniform
distribution of peptides, allowing for the sampling
of less fractions. In consequence, a reduction in
total measurement time of about 30% together
with a higher separation efficiency was achieved.
About 13 % more peptides and 7 % more proteins
could be identified with the alternative approach.
Further, the novel separation scheme prohibited
the strong salt loads typically for SCX, thus
increasing the robustness of the analytical platform
(1,2).
combination of both the advantages of the RP x
IP-RP-approach with the advantages offered by
offline-coupling of chromatographic separation with
MALDI TOF/TOF MS lead to a significantly
increased number of identified proteins (>150%)
compared to online-coupling to ESI IT MS. Upon
application of this novel analytical platform, more
than 50 % of all proteins theoretically expressed in
C. glutamicum could be identified. Besides high
abundant proteins, a number of low abundant
proteins, such as regulatory proteins, were found
(3). First results also indicate the suitability of the
method for quantitative measurements using the
iTRAQ strategy.
Innovative aspects
- Improved multidimensional separation scheme
- Increased robustness
- Complementary use of ESI and MALDI MS
References
(1) N. Delmotte et al., Two-dimensional reversed-
phase x ion-pair reversed-phase HPLC: an
alternative approach to high-resolution peptide
separation for shotgun proteome analysis. J.
Proteome Res., 6, 4363, (2007).
(2) N. Delmotte et al., Repeatability of off-line two-
dimensional chromatographic separations and
peptide identifications for shotgun proteome
analysis. Submitted (2008).
(3) M. Lasaosa et al., Shotgun proteome analysis
by two-dimensional reversed-phase x ion-pair
reversed-phase chromatography coupled to
MALDI TOF/TOF MS. Submitted (2008).
Contact address:
Andreas Tholey, Technische Biochemie –
Functional Proteomics Group, Saarland University,
66123 Saarbrücken, Germany
e-mail: a.tholey@mx.uni-saarland.de
 New MS-Based Serum Pattern Diagnostics method using ZipTip Technology and
Ultrafiltration
1 1 2 3 3 4 2
Ali Tiss , Celia Smith , John Timms , Zhiyuan Luo , Alex Gammerman , Usha Menon , Mike Waterfield , Ian
4 1
Jacobs , and Rainer Cramer .

1
The BioCentre Facility, University of Reading, Whiteknights, Reading RG6 6AS, UK.
Email: a.tiss@reading.ac.uk;
2
Cancer Proteomics Laboratory, Department of Gynaecological Oncology, University College of London,
Gower Street, London, WC1E 6BT, UK.
3
Dept of Computer Science, Royal Holloway, University of London, Egham TW20 0EX, UK
4
Institute of Women’s Health, UCL, Maple House, 149 Tottenham Court Road, London, W1T 7DN, UK.
peaks (more than 100 peaks per profile). The
results show that this easy and fast method of
using ZipTips, tested over a whole year, is more
Introduction reproducible and more suitable for serum profile
screening than magnetic bead-based
Blood metabolites and peptides are potential
methodologies used for comparison.
indicators of progression from a normal to a
diseased state. However, the presence of salts, An extra step of ultra-filtration was found to further
lipids and highly abundant proteins in blood serum increase the number and the intensity of low mass
can adversely affect sensitive proteomic analysis peptides (up to 6 kDa) without sacrificing the
using mass spectrometry. robustness of the technique. This last step also
Several pre-fractionation strategies using improved the MS/MS data acquisition.
chromatographic adsorbents have been employed The automated ZipTip platform is now used to
to desalt samples and remove abundant proteins. process thousands of serum samples collected
We describe here the adaptation and validation of from post-menopausal women as part of the UK
a simple and fast solid-phase extraction technique Collaborative Trial of Ovarian Cancer Screening
using ZipTips followed by ultra-filtration for peptide (UKCTOCS) trial.
serum profiling and identification.

Method Innovative Aspects

For method optimisation and comparison, we used • Robust, easy and sensitive method for
3 commercially available human sera and purification and enrichment of peptides
fractionated them using pipette tips packed with from biofluids, using ZipTips and ultra-
various solid phase materials. The sample filtration prior to biomarker pattern
preparations were run both manually and diagnostics using MALDI- or ESI-MS and
automatically. A CyBi™-Disk robot (CyBio AG) MS/MS.
equipped with a 96-piston head was adapted for
• Suitable method for use in a high-
automated sample preparation using ZipTips.
throughput and potentially clinical
Microcon centrifugal filters (Millipore) with various
environment, only requiring 5 to 10 μl of
cut-offs were used for the ultrafiltration either
serum.
before or after ZipTips.
MS data (from 700 to 10000 Da) were collected
using an Ultraflex MALDI-TOF mass spectrometer
(Bruker Daltonics). MS spectra were analysed
using FlexAnalysis and ClinProTools software
(both from Bruker Daltonics). MS/MS spectra of
the major peaks were collected using the MALDI-
QTOF Premier (Waters).

Results

Using standardised protocols and implementing a

multi-step quality control system, the average CV
of serum profiles within- and between-run
replicates has been evaluated and found to be
around 10% based on the variability of all detected
 New methodology combining hydrophobic gel and FT-ICR MS/MS for membrane
protein analysis

Caroline Tokarski, Marianne Fillet, and Christian Rolando

Chimie Organique et Macromoléculaire, UMR CNRS 8009, Protéomique, Modifications Post-traductionnelles
et Glycobiologie, IFR 147, USTL, 59655 Villeneuve d'Ascq Cedex, France

Introduction developed methodology are identified for the first

We propose the development of a method based time.
on new acrylamide gel improved for the separation
of hydrophobic proteins, and FT-ICR mass Innovative aspects
spectrometry. The separative strategy is based on • New hydrophobic matrix
the incorporation of the N,N'-dimethylacrylamide, • Identification of very hydrophobic proteins
in the composition of a classical gel, this monomer (example: peptide GRAVY value of 1.450)
increasing the hydrophobic reaction between the • Novel methodology combining home-made
gel matrix and the membrane proteins. The hydrophobic gel and on-line nanoLC nanoESI-
analytical part is based on the on-line nanoLC FT-ICR MS for membrane protein identification
nanoESI-Qq-FT-ICR-MS. The efficiency of this (example: ionic channel with 10-
new methodology was evaluated on membrane transmembrane- domains identified)
proteins of human colon HCT-116 cell line.

Methods
Membrane protein extraction from human colon
HCT-116 cell line was investigated using sodium
dodecyl sulfate-based buffer and the proteins were
separated on modified gels with the N,N'-
dimethylacrylamide monomer. Each gel was totally
sliced in 50 bands and each band was
enzymatically hydrolyzed. The peptides generated
by the trypsin hydrolysis are separated on a LC-
Packings nanoLC using a C18 trap-column (300
μm i.d., 5 mm length, Dionex) and a C18 capillary
column (Pepmap C18, 75 μm i.d., 15 cm length). Figure 1. HCT-116 membrane proteins separated on
classical and hydrophobic gels. The hydrophobic gel
The peptides are on-line transferred to the
presents higher resolving power than classical one.
nanoESI-Qq-FT-ICR MS instrument (Apex 9.4 T,
Bruker), separated and sequenced for protein
identification.

Results
A total of 227 proteins separated on hydrophobic
gel versus 195 on the classical gel were identified
(Fig 1). More hydrophobic proteins were globally
identified in the new gel (GRAVY -0.25) compared
Figure 2. MS/MS spectrum identifying the sequence
to the classical acrylamide one (GRAVY -0.42). 52
LAVEALVR corresponding to the peptide 194-201 of the
proteins were identified on the hydrophobic gel Hyaluronan synthase 1 protein (Accession number:
containing 1 to 10 transmembrane domains (vs 9 Q92839). The identified peptide is very hydrophobic, i.e.,
in classical gels). For example, the GRAVY value: 1.450. The Hyaluronan synthase 1 is a
sodium/potassium-transporting-ATPase-alpha-1- protein with 7 transmembrane domains located in the
chain-precursor (10-transmembrane-domains) or plasma membrane.
the glutamate-receptor (4-transmembrane-
domains) were identified only in the hydrophobic
gel with MS/MS mean errors inferior to 1 ppm.
Other proteins involved in
intracellular/extracellular-trafficking were identified
as the clathrin located onto the cytoplasmic side of
the plasma membrane. Focusing more particularly
on sequenced peptides, very hydrophobic peptides
could be observed, as the peptide 194-201 of the
Hyaluronan synthase 1 (Fig 2) identified despite its
very high GRAVY value of 1.45. Globally, most of
the membrane proteins identified with the classical
procedure are well documented in the literature
while membrane proteins strictly identified with the
 Selective detection and enrichment of serum albumin in human erythrocyte and urine by
capillary electrophoresis
1,3 1 2,3
W.-L. Tseng, C.-Y. Lin, H.-C. Chang
1. Department of Chemistry, National Sun Yat-sen University, Taiwan
2. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University
3. National Sun Yat-sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan

Introduction: Innovative aspects

Sodium dodecyl sulfates (SDS) is well known to • Selective detection and enrichment of HSA by
bind strongly to proteins and cause their partial-filled method in CE.
conformation change. High concentrations of SDS • The detection of HSA in human erythrocyte
are commonly used to unify their electrical charge and urine has been accomplished by CE
in capillary and polyacrylamide gel electrophoresis. coupled with universal absorbance detection.
Additionally, very little research has been devoted
to understanding the effect of alkyl chain length on
separation efficiency. It is noted binding constants
6 6
of 0.1 × 10 and 1.7 × 10 for octyl sulfate and
myristyl sulfate to bovine serum albumin,
respectively. Very little research has been devoted
to understanding the effect of alkyl chain length on
separation efficiency. Here, we introduced partial-
filling method for selective detection and
concentration of human serum albumin by capillary
electrophoresis with UV absorbance.

Methods:
Before separation, 70-cm capillaries were filled
with 1% dextran sulfate, which was prepared in 10
mM phosphate buffer at pH 10.0. A plug of
different concentrations of the sodium alkyl sulfate
solution was injected hydrodynamically by raising
Figure 1. On-line concentration of (A, B) 1 μM and
the capillary inlet 20-cm height for 10 s before
(C) 5 nM HSA by capillary electrophoresis (A)
proteins were injected by raising the capillary inlet
without and (B, C) with a plug of 1% SOS. The
20-cm height for a period of time up to 240 s. The
sample injection volume is 190 nL. Peak identifies:
ends of the capillary were then immersed in the
1, EOF marker; 2, HSA.
cathodic and anodic vials containing 1% dextran
sulfate (pH 9.0). Once a high voltage (17.5 kV)
was applied, the solution of dextran sulfate in the
anodic reservoir entered the capillary under
electroosmotic flow.

Results:
We have carefully investigated the effects that the
alkyl chain length on the electrophoretic mobility of
the proteins. An increase in electrophoretic
mobility was only discovered in the case of HSA
when applying a short plug of 1% sodium octyl
sulfate (SOS). The complete unfolding for HSA
occurs at 1% SOS. The formation of SOS-HSA
complexes shows at least 100-fold improvement in
peak efficiency. Our proposed method is
appropriate for on-line concentration of HSA.
Under injection a large-volume sample, the limit of
detection at signal to noise of 3 was 1.0 nM for Figure 2. On-line concentration of HSA in
HSA when using low cost absorbance detection erythrocytes by capillary electrophoresis (A)
coupled with capillary electrophoresis. With high without and (B, C) with a plug of 1% SOS. The
sensitivity and efficiency, the proposed method erythrocytes were spiked (A, B) without and (C)
has been shown for analyses of serum albumin in with 1 μM HSA.
human erythrocyte and urine.
 Ultrasonic Assisted Protein Enzymatic Digestion for Selenium amino-acids
1 4 2 3 2 1
G. Vale , R. Rial-Otero , A. Mota , L. Fonseca , M. L. Gonçalves , J.L. Capelo
1
REQUIMTE, FCT, Universidade Nova de Lisboa, Monte de Caparica, Portugal.
2 3
Centro de Química Estrutural, and IBB-Institute for Biotechnology and Bioengineering, Centre for Biological
and Chemical Engineering, from the Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
4
Nutrition and Bromatology Group, Analytical and Food Chemistry Department, University of Vigo, Ourense
Campus, E32400 Ourense, Spain
Introduction: Ultrasonic assisted protein Table 1 – Influence of sonication on enzyme activity
enzymatic digestion, USAPED, is a new sample (n=3)
treatment, for fast protein digestion. The
Applicationrelatively of USAPED accelerates, from Sonication Enzyme Enzyme activity
hours to minutes the sample treatment for: time (s) Activity (%)
a)identification of proteins by mass spectrometry (U/mg solid)
based techniques b) identification of amino acids 0 4.81±0.03 100
joined to metals such as selenium (Se-AA) [1,2]. 60 3.8±0.4 78±8
In this work the performance of USAPED for Se- 120 0.06±0.003 1.2±0.6
AA determination through hyphenated techniques
such as High Performance Liquid Chromatography Retention times –HPLC- for the different Se-AA
(HPLC) plus Electrothermal Atomic Absorption Species after enzymatic treatment
Spectrometry (ET-AAS)) is compared with HPLC-
MS/MS techniques. The retention times were studied using standards.
The enzymatic treatment with US seemed not to
Methods: change the integrity of the Se-AA species.
Each sample [Reference Materials BCR-414
plankton, ERM-CE 278 mussel tissue] was
prepared mixing 10 mg of the biological material
with 10 mg of enzyme in 1mL of ultrapure water.
The mixture was: 1) sonicated for 120s at 50%
sonication amplitude with a 1mm probe; 2)
centrifuged at 4000 rpm for 4 minutes and 3)
1 2 3 4
Passed through a 0,22μm Cut-off filter. 5
Hyphenated techniques: I) the extracts were
injected in an HPLC and fractions containing the
Se-AA, were recovered at retention times,
Figure 1 – Chromatogram of different Se-AA
previously determined with the adequate
mixture. 1 – Selenocystine ; 2- Selenio Methil Selenio
standards. II) the samples were analyzed for its Cysteine; 3- Selenite (Se IV); 4- Selenio Methionine; 5-
content in Se through ET-ASS. Selenate (SeVI)
HPLC-MS/MS: The extracts were injected in the
HPLC and the Se-AA, previously identified as USAPED arise as a robust fast sample treatment
targets, were further fragmented for Se containing for the proteomic applications of metallomics.
residue determination. Through this innovative sample treatment, other
techniques different from those using mass
Results spectrometry can be used for proteomic studies in
a routine basis, i.e. environmental and food
Influence of ultrasound in protease activity. control.
The influence of the ultrasonic energy in the Innovative aspects
enzymatic activity (casein hydrolysis) of protease
XIV was studied by applying ultrasound at • Acceleration of Se-AA extraction from solid
amplitude of 50% in intervals of 30–120 s. matrices
Interestingly, 30 s of sonication does not affect • USAPED-HPLC-ET-AAS Hyphenation.
enzyme activity, however, after 60 s, the activity
decreases ca. 20%. A sonication time 400of 120 s References
led to the complete inactivation of the enzyme [1] H. M. Santos, R. Rial-Otero, L. Fernandes, G.
(Table 1). Vale, M. G. Rivas, I. Moura, J. L. Capelo, J.
Proteome Res. 6 (2007) 3393.
[2] P. Bermejo, J. L. Capelo, A. Mota, Y.
Madrid and C. Cámara, Trac Trends Anal.
Chem., 23 (2004) 65.
 Potential of long capillary monolithic columns for the analysis of protein digests

Michiel van de Meent and Gerardus J. De Jong

Department of Pharmaceutical Analysis, Utrecht University,
Sorbonnelaan 16, 3584 CA Utrecht, the Netherlands

Introduction A B
Mass spectrometry of protein digests is an
important tool for protein identification in
proteomics. Analysis of protein mixtures requires
efficient separation of the peptides for optimal
protein identification, this can be achieved by using
monolithic columns. The gain in separation
efficiency for monolithic columns was evaluated by C D
the analysis of a mixture of bovine serum albumin,
α-casein and β-casein tryptic digests using an LC-
MS system and capillary columns of different
lengths (150 and 750 mm).

Methods
A protein digest mixture was separated by
Reversed phase liquid chromatography using
capillary monolithic silica columns of different
lengths at various gradient times. Both
chromatographic performance and efficiency of
protein identification were compared for both
columns. Peak capacities were determined from
MS base-peak chromatograms and MS/MS data
were used for protein identification by Mascot
database searching.
Figure 1:
Top figures – base peak chromatograms of digest
mixture. A: 150 x 0.1 mm column, 5-50% ACN (15 min)
gradient. B: 750 x 0.1 mm column, 5-50% ACN (75 min)
gradient.
Bottom figures – Averaged mass spectra of α-Casein
peptide YLGYLEQLLR (m/z 1267.7, MH+). C: 150 x 0.1
mm column, 15 min gradient. D: 750 x 0.1 mm column,
75 min gradient.

Results
Analyses with similar gradient slope for the two
columns produced ratios of the peak capacities
which were slightly higher than the expected value
of the square root of the column length ratio. Peak
capacity and protein identification scores were
higher for the long column, peak capacity ratios
varied from 2.58-3.23 for four different gradient
slopes, while protein identification scores were 1.3-
3.0 times higher. Only use of the longest gradient
on the long column led to identification of all three
proteins, which demonstrates the advantage of
increased separation efficiency. The use of long
monolithic columns improves peptide separation
and increases reliability of protein identification.

Innovative aspects
• Comparison of long and short silica monolithic
columns for peptide separation
 REPRODUCIBILITY OF 2D GEL-BASED PROTEOMICS EXPERIMENTS
1 2 3 4 5 6
David Bramwell , Mary Caponite Hurley , Alamgir Khan , Katrin Marcus , Jules A. Westbrook , Hans Voshol
6
Novartis Inst. for BioMedical Research, WSJ-88.805, CH-4002 Basel, Switzerland

Introduction achieve an equivalent level of confidence in the

A key concept in an experimentally driven scientific results obtained with that approach.
discipline such as proteomics is the notion that
results generated in one facility can be reliably
reproduced in another. Because of the inherent

Correlation coefficient
complexity of ‘the proteome’, reproducibility
continues to be an important issue in proteomics
[1]. Consequently, proteomic studies have not yet
resulted in the once anticipated quantum jump in
the discovery of validated disease targets and
biomarkers. Here we demonstrate the validation of
2D-PAGE as a reproducible approach for
differential proteome analysis through a cross-lab
1 2 3 4 5
experiment.
Participating labs
Methods
Figure 1. Intra-lab reproducibility - whole gel
The experiment was designed to meet one key correlation.
goal: perform a 2D gel-based comparison between Average whole gel correlation coefficients (Pearson),
two biologically relevant, complex samples and within labs but across multiple experiment runs and
reproducibly identify differential spots. control/treated conditions: 0.92 (lab 1), 0.95 (lab 2), 0.86
Haemophilus influenzae, a Gram-negative (lab 3), 0.87 (lab 4), 0.89 (lab 5). The first submitted gel
bacterium, was treated with actinonin, a peptide from each lab was used as a reference for that lab.
deformylase inhibitor [2]. Control and treated
samples were extracted and distributed to 5 expert
labs, where they were separated by 2D PAGE Innovative aspects
using a procedure that was kept constant within • Validation of 2D PAGE at the level of the final
the instrumental restrictions of the respective analysis result
laboratories. Participants were asked to identify • Basis for establishing standard samples and
the 200 most significant differences using protocols for 2D PAGE
Progenesis SameSpots software. Images were • Paradigm for other proteomics approaches
also provided to a third party for an independent such as LC-MS
analysis. After completion, a meta-analysis was
carried out to compare the identified differentials. References
1. Rifai N, Gillette MA, Carr SA. Nat. Biotech.
Results 2006, 24, 971.
Participants provided at least one experiment, 2. Bandow JE et al., Proteomics, 2003, 3, 299.
defined as 5 replicate gels of each sample, with
some labs including multiple repeats. Intra-lab Author affiliations:
1
reproducibility was excellent for each of the Nonlinear Dynamics, Newcastle upon Tyne NE1
datasets, as illustrated by whole image correlation 2ET, UK
2
coefficients of 86 - 95% (Fig. 1), with scatter plots Michigan Proteome Consortium, Ann Arbor, MI
of spot volumes showing standard deviations 48109-0404, USA.
3
below 0.09. Inter-lab parameters were equally Australian Proteome Analysis Facility (APAF),
impressive, allowing a significant number of gel Macquarie University, Sydney NSW 2109
images to be aligned at the pixel level across labs. Australia.
4
Most importantly, lists of differential spots were Medical Proteom-Center, Ruhr-University of
highly consistent across all participating labs, Bochum, D-44801 Bochum, Germany.
5
demonstrating that 2D PAGE is a reliable Proteome Research Centre, UCD Conway
approach for the identification of differentially Institute of Biomolecular and Biomedical
expressed proteins. Research, University College Dublin, Ireland
In conclusion, this study validates 2D gel-based
comparative analysis by reproducibly deriving tens
to hundreds of differentially expressed proteins
across labs. We highly recommend repeating a
similar experiment in an LC-MS setting, in order to
 An Integrated M-IPG-CIEF-microenzymatic reactor-CZE platform for protein analysis

Tingting Wang, Zhen Liang, Lihua Zhang, Yukui Zhang

Devision of Biotechnology, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, Dalian 116023, China

Introduction Innovative aspects

Although 2D-PAGE has played extremely • Monolithic immobilized pH gradient CIEF as
important role in proteome study, it still suffers the first dimensional separation.
from disadvantages for the separation of extremely • Integration of 2D-CE with on-line
acidic and basic proteins, and solid phase based microenzymatic reactor.
operation. Therefore, in recent years, much effort
has been made on 2D-capillary electrophoresis References
(CE) to provide complementary platforms. (1) N. J. Dovichi et al, CE-Microreactor-CE-
Recently, Dovichi et al integrated the intact MS/MS for Protein Analysis; Anal. Chem.
proteins separation first by CSE, on-line digestion 2007, 79, 2230-2238.
by pepsin immobilized microreactor, the separation
of resulting peptides by MEKC and the protein
identified by MS/MS, and showed the promising of
the combination of top-down and bottom-up
strategies (1). In our recent work, a novel CE
based integrated platform for protein study was
established.

Methods
As shown in Figure 1, an integrated 2D-CE Figure 1. Schematic of the Integrated M-IPG-CIEF-
microenzymatic reactor-CZE platform.
platform was established with monolithic
immobilized pH gradient (M-IPG) CIEF for protein
separation, trypsin immobilized microenzymatic
reactor for on-line protein digestion, and CZE for
peptide separation via two proper interfaces. The
hollow fiber membrane interface between M-IPG
CIEF and microenzymatic reactor was used for the
adjustment of buffer pH to improve the
compatibility of protein separation and digestion.
The Teflon tubing interface between
microenzymatic reactor and CZE was used to
change the buffer to that suitable for peptide
separation and introduce an electric field for CZE
as well.

Results
With such an integrated M-IPG CIEF-
microenzymatic reactor-CZE platform, proteins
were hydrodynamically introduced into the M-IPG
column by a syringe pump and off-line focusing
was performed. Subsequently, the separated
proteins were transferred to the microreactor by a
syringe pump at the flow rate of 500 nl/min for 1.5
min. Finally, the resulting peptides from each
fraction were separated by CZE in 12 min. The
performance of such a system was demonstrated
by the analysis of myoglobin. In 7 of 12 fractions of
protein separation, peptides were observed by
CZE, and a total peak capacity of 2000 was
obtained. Further work on the analysis of complex
protein samples by such a platform is undergoing,
and the primary data indicates that it might offer a
useful way for proteome study.
 Quantitative Proteomics for 2D Gel Electrophoresis using Multiple Saturation Dyes
1,2 2 1,2 1
John E. Wiktorowicz , Susan Stafford , Alexander Kurosky , Dept. Biochemistry and Molecular Biology ,
2
and the Biomolecular Resource Facility , The University of Texas Medical Branch, Galveston, TX, USA

Introduction 2. Galvani, M., Rovatti, L., Hamdan, M., Herbert,

Quantitative approaches for proteomics involve
labeling with stable isotopes or fluorescent dyes.
Both require stringent chemistry and residue
targets to minimize non-specific modifications for
accurate quantification, and true saturation for
reproducibility. Protein mixture complexity, and
extraction buffers that include urea, thiourea, and
detergents that interfere with labeling, further
confound quantitative analysis. We have
investigated the use of uncharged, inexpensive
fluorescent dyes targeting protein thiols under truly
saturating amounts and in the presence of
confounding buffer components.
B., and Righetti, P. G. (2001) Protein alkylation
in the presence/absence of thiourea in
proteome analysis: a matrix assisted laser
desorption/ionization-time of flight-mass
spectrometry investigation, Electrophoresis 22,
2066-2074.
3. Tyagarajan, K., Pretzer, E. L., and Wiktorowicz,
J. E. (2003) Thiol-reactive dyes for fluorescence
labeling of proteomic samples, Electrophoresis
24, 2348-2358.

Methods
Cysteine content of standard or tissue extracted
proteins was estimated by amino acid analysis.
Sufficient dyes (BODIPY FL- and BODIPY 577-
maleimide) were incubated with the cysteine-
containing and cysteine-free (as negative control)
proteins under variable conditions as described
below. Gels were imaged for fluorescence and
then stained for total protein with Coomassie.
Where appropriate, specific fluorescence (SF) was
calculated as the ratio of the sum of pixel
intensities of each fluorescent protein band and
the corresponding Coomassie band minus the SF
of the non-cysteine containing protein. Both
intensities were background corrected.

Results
Labeling in thiourea with iodoacetamides versus
maleimides confirmed the observations of
iodoacetamide inhibition (2, 3). Other conditions,
such as the linearity of labelling, the effect of dye
concentration, pH, and reaction time on
completeness and specificity of labeling were
optimized, and the impact on protein mobility, and
the effect of additional, unconventional additives
were assessed. In addition, the sensitivity,
reproducibility, and quantitative accuracy were
also investigated and will be presented.

Innovative aspects True saturation (dye:protein

thiol > 50-fold)
• Uncharged dyes with no impact on 2D gel
electrophoresis migration
• Accurate, reproducible, sensitive, and
inexpensive fluorescence labeling

References
1. Pretzer, E., and Wiktorowicz, J. E. (2008)
Saturation fluorescence labeling of proteins for
proteomic analyses, Analytical Biochemistry
374, 250-262.
 High Sensitivity Porous Sprayer (HSPS) in Capillary Electrophoresis-Mass
Spectrometry (CE-MS) for the Analysis of Phosphopeptides.

1 2 2
C. C. L. Wong , The Scripps Research Institute C. Ratnayake , J. Chapman , Beckman Coulter, Inc. J. R.
1
Yates III , The Scripps Research Institute.
1
The Scripps Research Institute, La Jolla, CA 92037, USA 2Discovery Products, Beckman Coulter, Inc., Fullerton CA,
USA

Introduction: Protein phosphorylation generally

occurs at low levels and increases the hydrophylicity of exclusion settings were used to capture fast moving
peptides. In reversed-phase liquid chromatography closely resolved peptides.
these peptides show less binding affinity for the
hydrophobic columns, thus makes the characterization U:\catclw\...\22908Run4_080229104110 2/29/2008 2:48:45 PM

become challenging. In capillary electrophoresis, the RT: 0.00 - 66.54

100
26.51
27.88
NL:
1.20E7
B ase P eak

Relative Abundance
80 MS
28.06
26.32 22908Run4_

low isoelectric points of phosphopeptides were utilized 60 28.70 080229104110

25.05 29.26
40 32.49
34.30
24.24
20
23.18 30.18

to separate them from non-phosphopeptides thus

2.00 4.07 5.53 7.31 8.92 14.17 16.39 17.67 19.13 35.24 38.98 41.91 44.91 46.20 51.02 52.84 56.75 59.44 63.47
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
T im e (m i n)

reducing the complexity of the peptide mixture. In this

RT: 19.72 - 36.99
26.51 NL:
100 1.20E7
27.88
27.18 B ase P eak

Relative Abundance
80 MS
28.06 28.36
26.32 22908Run4_
25.51 28.70

study, a high sensitivity porous sprayer was developed

60 080229104110
25.05 29.11
24.63 29.32
40 32.49
32.38 34.30
24.24 29.38 34.38
20 34.23
23.18 24.20 32.56 34.60
19.99 20.73 21.42 22.31 23.07 29.51 30.18 30.72 31.82 34.05 35.24 35.54 36.97

as an interface of CE-MS and achieved very low

0
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
T im e (m i n)

RT: 0.00 - 66.54

detection limits for phosphopeptides. Relative Abundance 100

80

60
23.18 N L:
9.50E5
B ase P eak m/z=
1031.33-1032.33
MS
22908Run4_0802
29104110
40
25.91
20 27.88 29.46
32.49 35.18 36.97 52.84
0.19 3.93 4.84 6.42 8.61 10.85 12.11 15.25 19.28 22.33 41.52 41.98 44.05 47.57 51.02 53.57 58.44 59.77 60.81 65.03

Methods: PA 800 Protein Characterization System

0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
T i me (m i n)

22908Run4_080229104110 #7004 RT : 23.18 AV: 1 NL: 9.50E5

(Beckman Coulter) and LTQ XL (Thermo Fisher

T : ITMS + c ESI Ful l ms [150.00-2000.00]
1031.89
100
Relative Abundance

80

Scientific) instruments and fused silica capillaries, 30

60
701.04
40
176.82
1043.31
20 370.98 445.13
216.93 279.08 532.17 696.21 709.15 839.58 938.34 976.71 1062.81 1204.37 1273.90 1376.10 1497.70 1548.41 1712.16 1864.93 1931.08
0

um ID and 150 um OD (Polymicro Technologies) were 200 300 400 500 600 700 800 900 1000 1100
m/z
1200 1300 1400 1500 1600 1700 1800 1900 2000

used for all CE-MS experiments. Capillaries were

etched with 49% HF to obtain 3-4 cm long porous tip. Figure 1: Full CE-MS chromatogram of β-casein digest
All the CE capillaries were conditioned and coated to (A), enlarged region (B), extracted chromatogram of this
generate a positively charged layer. Etched capillary phosphopeptide K.FQS*EEQQQTEDELQDK.I
was simply inserted into the specially designed spray (precursor mass: m/z 1031.83, charge 2+),
correspondent spectrum of this peptide (D)
needle to connect the CE capillary to MS instrument
and the needle was filled with 1:1 methanol: water and
0.1% acetic acid mixture for electrical contact.
Neurotensin, methoxamine and beta-casein digest were
used as analytes. Concentration of formic acid and
acetonitrile were adjusted to optimize the CE-MS
condition.

Results: A robust novel sheath less CE-MS ESI

interface that is built in to the CE separation capillary
was used to achieve a high sensitivity, stability and ease
of use. The electrical connection to the capillary outlet
was achieved through the porous tip prepared by
etching the tip of separation capillary with hydrofluoric
acid, significantly eliminate the bubble formation due to
redox reactions at the electrodes when high voltage was
applied. The flow rate the capillary was maintained Figure 2: SEQEST result of β-casein typtic digest using
around 100 nl/μL constantly. Hydrophobic interaction CE-MS
of peptides with the capillary wall was minimized by
hydrophilic coating. Stable flow at low pH (~3.0) was
maintained due to positive charge on the capillary wall.
The detection limit of neurotensin and methoxamine
was 103 fold lower using HSPS, compared to the
conventional sheath liquid interface. Beta-casein typtic
digests were used as model protein for detection of
phosphopeptides. Low abundant phosphopeptides were
detected at the concentration of 10 ng/uL. High peak
capacity gained by CE separation was maintained in the
CE-MS interface. Optimized data dependant dynamic
 Single drop microextraction using Ag nanoparticles for peptide analysis in AP-MALDI-MS

Putty-Reddy Sudhir1,2, Zi-Cong Zhou1,2 and Hui-Fen Wu3*

1Department of Physics, Tamkang University, Tamsui, 251, Taiwan

2Graduate Institute of Life Sciences, Tamkang University, Tamsui, 251, Taiwan

3Department of Chemistry, National Sun Yat - Sen University, 804, Kaohsiung, Taiwan

Introduction and proteins via hydrophobic interactions [2].

Metallic nanoparticles have been intensively However, the weak hydrophobic interactions take

applied as affinity probes in sample preparation for a long time (1.5hour) for extraction. Therefore, in

proteins or other biomolecule analysis in mass this study, we applied the electrostatic approach of

spectrometry. However, these nanoparticle based siliver nanoparticles via SDME technique in the

affinity techniques require the separation or separation and preconcentration of hydrophilic

washing of the nanoparticles from protein sample peptides from sample solution before MALDI-MS

before mass spectrometry analysis. The analysis in order to compare with our previous

separation or washing of nanoparticles after methods. The extraction parameters including

extraction may lead to serious sample losses and extraction time, sample pH and agitation rate were

chance of interference of matrices from samples optimized for best extraction efficiency and

because these techniques prepared the nano sensitive detection of peptide mixtures. In addition,

particles in aqueous phase. Thus, the application the applicability of the proposed method has been

of these techniques to low concentration of illustrated in the determination of gramicidin from

biomolecule analysis from biological matrices such biological samples and in high concentration of

as urine and blood is difficult. Therefore, in 2005, matrix interferences such as urea and Triton X-

we first reported the application of Single drop 100.

microextraction (SDME) method using

tetraalkylammoniumbromide capped gold Methods

nanoparticles prepared in toluene as electrostatic SDME experiments were perormed follow exactly

probes for rapid analysis and signal enhancement the same procedures from our previous study [1].

of peptide mixtures in the AP-MALDI atmospheric Briefly, the stock solutions of peptides were

pressure- matrix assisted laser prepared in water with a concentration of 1 μg/μL.

desorption/ionization mass spectrometry (AP- Peptides with desired concentrations were spiked

MALDI/MS) [1]. This approach is simple and into a glass vial filled with 20 mL sample solution.

samples can be directly deposited on target plates A 10 μL of microsyringe (Hamilton co., Reno,

for MALDI-MS analysis after extraction. In 2008, Nevada, USA) was taken and 2 μL toluene

we continually applied modified silver containing AgNPs was drawn into it. The

nanoparticles (AgNPs) with hydrophobic ligands microsyringe was inserted into sample solution

including dodecanethiol (DT) and octadecanethiol through the PTFE-coated silicon septum of screw

(OT) prepared in toluene via liquid-liquid cap of glass vial as shown in Fig. 1. As soon as

microextraction (LLME) to analyze neutral peptides the sample was extracted into 0.8 μL of droplet of
organic solvent or silver nanoparticles in toluene, The AP-MALDI/MS spectra of Leu-enk, and Met-

the droplet was drawn back into the microsyringe enk mainly show two protonated molecules at m/z

(Hamilton Co., Reno, NV) and directly placed on to values 556, 574 for Leu- and Met-enk,

the target plate for subsequent AP-MALDI/MS respectively. The AP-MALDI/MS of GrD spectra

analysis. Mass spectrometry experiments were mainly appear three ions at m/z 1883, 1906 and

carried out using a Finnigan MAT ion trap mass 1922 from GrA which were assigned [Val-GrA+H]+,

spectrometer (Finnigan LCQ-Advantage, San [Val-GrA+Na]+ and [Val-GrA+K]+, respectively.

Jose, CA, USA). The ESI ion source was replaced Gramicidins are neutral peptides and therefore

with an AP-MALDI ion source. Matrix solution was exhibit no charge at any pH. Leu-enk and Met-enk

prepared by using CHCA (10 mg/mL) dissolved in show net negative charge above the pI value since

methanol and water (2:1 ratio), then TFA was their pI value is 5.4. Based on the neutral property

added to the solution with a final concentration of of gramicidins and pI dependent charge exhibition

0.1%. Following the dried droplet method, 2 μL of of Leu- and Met- enkephalins, the following

nano silver extraction solution was placed on experiments were performed to demonstrate the

target plate with the addition of equal volume of application of AgNPs for peptide identification.

matrix solution and allowed to dry. Laser power

attenuated to 60% was used for the illumination at Effect of selection of solvent
10 Hz of repetitions. Capillary temperature was Several organic solvents including hexane,

maintained at 250 oC. 1.8 kV voltage was applied chloroform and toluene which are typical solvents

to the target plate. Capillary voltage and tube lens applied in traditional SDME experiments were

offset voltage were applied with 40V and 70V, examined for the extraction of the Leu- and Met-

respectively. A pulsed nitrogen laser with UV enkephalin peptides. The SDME were performed

radiation of 337 nm was used to desorb and ionize with extraction time of 2 min and agitation rate at

the deposited molecules. Each spectrum was 240 rpm. Met- and Leu- enkephalin peptides were

scanned for 2 min. However, any difference was spiked into aqueous solution with equal

not observed between 2 and 3 min of scanned concentration (3 μM). After extraction, the analysis

spectra. of liquid drop of the abovementioned solvents in

AP-MALDI/MS did not exhibit the signals

Results corresponding to the selected enkephalin peptides.

Parameters optimization for SDME and peptide The results only two ions of m/z 568 and 524.1

analysis by AP-MALDI/MS were observed. The MS/MS study of these peaks

The SDME extraction of peptide mixtures from revealed that they are coming from the CHCA

aqueous solution to organic solvent was influenced matrix. The m/z 568 is assigned as [3CHCA+H]+

by several parameters including solvent selection, and m/z, 524 is identified as [3CHCA-CO2+H]+.

extraction time, agitation rate and sample pH. The spiked concentration of the peptides was

Thus, these factors were optimized for all the increased to 12 μM and SDME was performed

SDME experiments. To evaluate this technique, a again with various organic solvents as mentioned

mixture of peptides containing gramicidin D (GrD), above. However, after extraction, the subsequent

Leu-enkephalin (Leu-enk) and Met-enkephalin AP-MALDI-MS analysis of all organic solvents can

(Met-enk) were selected as model compounds. not successfully to extract any enkephalins since

GrD consists of a group of different types of these organic solvents are nonpolar in nature and

gramicidin peptides known as GrA, GrB and GrC. therefore can not extract the polar enkephalin
peptides. Further, we applied SDME with toluene adding 0.1 N HCl and observed no signal

containing AgNPs to extract these peptides from corresponding to the peptides. The increase in the

aqueous solution since it is believed that pH to 9.0 by adding 0.1 N NaOH to the sample

nanoparticles exhibit surface charge and both Leu- solution obtained similar results as observed at pH

and Met-enkephalins were extracted and detected 7.0. The two model peptides were successfully

from the AP-MALDI/MS analysis. The Leu- and extracted from aqueous solution to AgNPs in

Met-enkephalins exhibited protonated molecules at toluene droplet at the pH values above their

m/z 556.2 and 574.1 respectively. These results isoelectric point (pI 5.4.) as the peptide molecules

reveal that AgNPs used for SDME exhibit charge exhibit net negative charges. Below the pI values,

on their surface to capture the peptides via these two peptides carry net positive charge and

electrostatic attraction forces. were thus kept away from positively charged

tetraalkylammonium ions located on the surface of

Effects of extraction time AgNPs. Furthermore, to demonstrate that only

To optimize the best extraction efficiency for charged species can be extracted by AgNPs,

extraction time of sample solution for this mixtures of neutral peptides (GrD) with Leu- and

technique, SDME using AgNPs liquid droplet was Met-enk were spiked into sample solution and

carried out with different time intervals from 30 sec SDME coupled with AP-MALDI/MS analysis was

to 7 min. The signal was increased with increasing performed at different pH values with a range from

time from 30 sec to 2 min and then decreased 5.0 to 11.0 again. However, AP-MALDI/MS

since the equilibrium between the peptides and analysis of the toluene droplet containing AgNPs

AgNPs has been reached at 2 min. These results did not show any signal for gramicidin peptides,

suggest that the equilibrium of peptides between which confirmed that peptides containing pI and

aqueous solution and the droplet of AgNPs in exhibit charge at a certain pH only were captured

toluene was extremely fast due to the electrostatic by AgNPs and subsequently identified by AP-

interaction. Therefore, we selected 2 min as the MALDI/MS. This result indicates that this

optimum extraction time for both enkephalin technique is based on the surface charge of

peptides. AgNPs as well as net charge of the peptides since

in our previous study, the gramicidin can be easily

The effect of sample pH extracted by the AgNPs coated with which is a

The effect of pH was studied to optimize the hydrophobic affinity probe.

sample pH conditions and to understand the

process of how the peptides are extracted into the Effects of stirring rate
droplet of AgNPs in toluene by electrostatic To optimize the extraction efficiency, the agitation

interactions. The SDME extractions were of sample solution was carried out with varying

performed at both acidic and alkaline from pH 5.0 stirring rates ranging from 60 - 360 rpm at different

– 11.0. The pH effect on signal intensity was intervals. Maximum extraction efficiency was

shown in Fig. 4B. The pH of the aqueous solution observed when the sample solution was agitated

was adjusted with 0.1 N HCl and 0.1 N NaOH. The at 240 rpm and then the extraction efficiency was

SDME/AP-MALDI/MS technique was successfully decreased with increasing agitation rate (300 and

applied to identifie both Leu- and Met-enkephalins 360 rpm), which is due to the instability of the

from aqueous sample solution with pH 7.0. The pH microdroplets at higher stirring rate (300 and 360)

of the sample solution was dropped to pH 3.0 by

rpm. Therefore, we selected 240 rpm as the best 100 or 6.0 M urea and subsequently identified by

sample agitation rate for the extraction of peptides. AP-MALDI/MS analysis as in Fig. 5B and Fig. 6,

respectively. Above the concentrations of 6.5 M

Detection limits of SDME-AgNPs/AP-MALDI/MS urea and 1.2% Triton X-100, the extraction

technique in aqueous samples efficiency and signal intensity were decreased

By means of optimized conditions, (2min, 240 rpm rapidly as the droplet of toluene containing AgNPs

and pH 7) the peptide mixtures of Met- and Leu- became unstable. In addition, since the AP-

enkephalins were identified at lower MALDI/MS with excellent tandem mass capability,

concentrations with the detection limits for Met- the identification of peptides in presence of high

and Leu-enkephalins for 160 and 210 nM matrix interference such as 1% Triton X-100 and

respectively. 6.0 M urea can be further characterized by using

tandem mass spectrometry (AP-MALDI/MS/MS).

Application of SDME-AgNPs/AP-MALDI/MS The MS/MS results of protonated molecule of Met-

technique in high interference samples enk (m/z, 574.1) produced several fragment ions

One of the problems experienced in MALDI-MS including [Met-enk-H2O+H]+ (m/z 556), b4 (m/z

analysis of protein samples is suppression of 424.93), a4 (m/z 397.01), y3 (m/z 353.91), and b3

signal intensity by surfactants such as Triton X-100 (m/z 424.93). The MS/MS spectra of protonated

and urea. Triton X-100 is a commonly used molecule of Leu-enk (m/z, 556.0) generated

surfactant for hydrophobic membrane proteins fragment ions including [Leu-enk-H2O+H]+ (m/z

solubulization during biological sample preparation 538.05), b4 (m/z 424.93), a4 (m/z 397.01), y3 (m/z

whereas urea is useful to denature the proteins 335.87) and b3 (m/z 278.88) ions. These observed

ahead of tryptic digestion in proteomic MS/MS results are consistent with those results

approaches. However, the presence of Triton X- observed in previous studies. These results

100 and urea in the samples dramatically affects revealed that this technique is a powerful tool for

the quality of mass spectra for subsequent protein peptide identification at high interfercnce matrix

identification when concentrations exceeding 0.1% even at the concentrations of 1% Triton X-100 and

and 1M of Triton X-100 and urea, respectively. To 6 M urea, which are sufficient for solubulization of

overcome this problem, an application has been hydrophobic membrane proteins and protein

demonstrated using AgNPs-assisted SDME denaturation.

coupled with AP-MALDI/MS/MS method. We first,

tried to identify 3 μM of both Met-enk and Leu-enk Comparison of mass spectra obtained from silver
peptides from aqueous solution in the AP-MALDI- and gold naoparticle-assisted SDME coupled with
MS analysis of sample solution containing either AP-MALDI-MS
1% of Triton X-100 or 6.0M of urea without SDME We previously reported the use of modified gold

and did not observe any peaks related to both enk- nanoparticles assisted with single drop

peptides in the AP-MALDI mass spectra. The microextraction (SDME) in the separation and

mass spectrum obtained without SDME analysis of preconcentration of these peptide mixtures from

sample solution containing urea was not shown in sample solutions before AP-MALDI/MS analysis

this article since no signal was obtained. However, [1]. This is because AuNPs prepared in toluene

the two peptides were successfully extracted by exhibits positively charged tetraalkyammonium

means of AgNPs-assisted SDME from exactly the ions on their surface and therefore exhibit net

same of sample solution containing 1% Triton X- positive charge [1]. In this study, we compared this
similar approach by changing the AuNPs to AgNPs. neutral peptides and proteins by hydrophobic

Thus, the AgNPs exhibit positive charge on their interactions [2]. The LODs of gramicidin detected by

surface and this is due to the interaction between using this approach in urine and plasma samples

dispersion medium (toluene, dodecanethiols, was 0.13 and 0.16 μM, respectively. Comparison of

tetraalkylammonium bromides) and AgNPs, that AgNPs acting as an electrostatic affinity probe

provoke AgNPs to act as electrostatic affinity (current approach) with AgNPs acting as a

probes for peptides. The mass spectra obtained hydrophobic affinity probe (previous work) [2] for

from AuNPs as well as AgNPs-assisted SDME peptide analysis in the AP-MALDI/MS, the AgNPs

techniques were compared and the main as electrostatic affinity probe is suitable to analyze

difference between them is that in our previous neutral peptides such as gramicidin while the

study with AuNPs-assisted SDME, the AP- current AgNPs was further stabilized by

MALDI/MS shown higher signal intensity for Leu- tetraalkyammonium ions which exhibits positively

enk than Met-enk. To our surprise, in contrast to charged ions on their surface and therefore we can

AuNPs, in exactly the same conditions such as in analyze peptides or proteins by electrostatic

aqueous sample solution, in presence of 1% Triton attraction forces based on controlling the pH of

X-100 or in the 6.0 M urea, all mass spectra solution. In addition, since the electrostatic

obtained with AgNPs exhibited higher signal interaction is much stronger than the hydrophobic

intensity for Met-enk although equal concentration interaction, we found that the extraction time can be

of peptides were spiked into sample solution. The greatly reduced in this current approach using

possible reason for the higher efficiency of AgNPs SDME-AgNPs (2 min) compared with that of our

towards Met-enk could be due to silver is highly previous LLME-AgNPs method (1.5 hour).

reactive with sulfur peptides where Met-enk

consists of sulfur atom. A recent report also Approach of using silver naoparticle prepared in
demonstrated that the most attractive sites for toluene as matrix for AP-MALDI ion trap mass
AgNPs would be the sulfur containing residues of spectrometry for peptide analysis
the glycoproteins of HIV-1. The above results AgNPs prepared in aqueous phase have been

show that AgNPs are more efficient to extract Met- successfully applied as matrix for peptide analysis

enk peptide relative to AuNPs. Another advantage in the MALDI –TOF- MS. Thus, we also examined

of using AgNPs in SDME is that the cost for this AgNPs as matrix in the AP-MALDI/MS.

AgNPs-SDME method is less expensive when Unfortunately, none of signal can be observed. Up

compared with AuNPs. Thus, it is worthy and also to date, none of the report has been shown that

of interest to introduce AgNPs for peptide any the nanoparticles can be applied as matrix in

identification. the AP-MALDI/MS. The reason may be attributed

to the poor sensitivity of AP-MALDI/MS.

Comparison of AgNPs acting as an electrostatic

affinity probe with AgNPs acting as a hydrophobic Innovative aspects
affinity probe for peptide analysis in the AP- z This technique provides basement to establish

MALDI/MS the Ag nanoparticle based methods for

We previously using modified AgNPs with purification and identification of proteins and

hydrophobic ligands including dodecanethiol (DT) peptides especially biomarkers in biological

and octadecanethiol (OT) prepared in toluene via samples.

liquid-liquid microextraction (LLME) to analyze

z This technique also brings together nano- and

bio-sciences especially in proteomics field.

z The SDME-AgNP technique is simple, rapid

and single step method for preconcentrating

and affinity probes for analysis of low

abundance of peptide mixtures in complex

matrices. In addition, this approach could be

efficiently preconcentrated samples without

sample loss prior to AP-MALDI/MS for

sensitive and analysis of peptides in biological

samples.

References
(1) Sudhir, P.R., Wu, H.F., Zhou, Z.C., Anal

Chem. 2005, 77, 7380-7385.

(2) Shrivas, K., Wu, H. F., Anal. Chem. 2008,

80 (in press).

Figure 1. Schematic representation of SDME-

AgNPs coupled with APMALDI/MS technique.

 Miniaturized two dimensional capillary electrophoresis on microchip for protein
analysis

Yongzheng Cong, Lihua Zhang, Yu Liang, Weibing Zhang, Yukui Zhang

National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China

Introduction • A 2D-CE on microchip platform was

Recently much effort has been made to perform established for the high throughput and high
multidimensional separation on microchip, in which resolution analysis of proteins.
different orthogonal separation mechanisms are • Sample zones focused in the first dimensional
employed. Microchip based multidimensional channel could be easily controlled and
separation might play an important role in the completely transferred to the second
analysis of complex samples, such as proteome. dimension without any loss
Here we describe a two dimensional capillary
electrophoresis platform on microchip with the References
combination of isoelectric focusing (IEF) and (1) Rocklin, R. D., Ramsey, R. S., Ramsey, J. M.,
capillary zone electrophoresis (CZE) for protein Anal. Chem. 2000, 72, 5244-5249
separation. (2) Herr, A. E., Molho, J. I., Drouvalakis, K. A.,
Mikkelsen, J. C., Utz, P. J., Santiago J. G., Kenny,
Methods T. W., Anal. Chem. 2003, 75, 1180-1187
Glass chips were fabricated by standard (3)Yongzheng Cong, Lihua Zhang, Yu Liang,
photolithography and wet chemical etching with Dingyin Tao, Weibing Zhang, Yukui Zhang, J. Sep.
the channel 100μm wide and 40μm deep (Fig 1). Sci. 2008 (in press)
With polyacrylamide as the permanent coating, the
-
EOF was suppressed, and decreased to 3.4×10
9 2
m /Vs. During the separation, proteins were first
focused by IEF in the first dimensional separation,
and then directly driven into the second
dimensional channel by controlling the applied
voltage though chemical mobilization, followed by
the separation by CZE.

Results
Effects of various experimental parameters, Figure 1 The layout of the 2-D microchip
including the electric field strength, channel length electrophoresis.
and injection frequency from the first to the second The first dimension separation (IEF) was performed in
dimensional separation channel, were studied. It the channel between reservoirs 1 (anolyte) and 2
was found that the increase of electric field (catholyte); the second dimension separation (CZE) was
strength was helpful to enhance the resolution of performed in the channel between reservoirs 3 and 4.
the separation, and. a longer channel length could
lead to higher resolution of CZE. The focused
sample zones were introduced into the second one
in sequence by switching the voltage. Smaller
volume of sample introduced lead to higher peak
capacity of the 2-D system.
Under the optimized condition, the peak capacity
of 795 was obtained for the separation of three
labeled proteins, which was greatly increased
compared with each single dimensional
separation. The capacity of such a platform was
further demonstrated by analyzing proteins Figure 2 Analysis of proteins extracted from E. Coli by 2-
extracted from E. Coli (Fig 2), and All these results D microchip electrophoresis
showed the promising of multidimensional Experimental conditions: IEF: cathode solution, 20 mM
separation on microchip in the high throughput and NaOH containing 0.2% MC; anode solution, 20 mM
high resolution analysis of complex samples. H3PO4 containing 0.2% MC; separation voltage,
1300v/cm; CZE: buffer: 20 mM H3PO4 (pH=2.1)
Innovative aspects containing 0.2% MC; injection and separation voltage,
3oov/cm and 500v/cm; sampling and separation time: 4s
and 56s. Sample: 1mg/mL extracted proteins in solution
of 1.0 % MC and 2% v/v Pharmalyte. Image b was the
processed planer images corresponding to a
Column switch recycling size exclusion chromatography for high
efficientt protein separation
efficien
Huiming Yuan 1,2, Lihua Zhang 1, Zhen Liang 1, Yukui Zhang 1
(1 National Chromat ographic Reasearch and Analysis Center, Dalian Institute of Chemical
Physics, The Chinese Academy of Sciences, Dalian ,P.R.China. 2Graduation university of
chinese Academy of Sciences, Beijing, P.R.China)
Introduction orthogonally integrated with RPLC. With a
Due to the good biocompatibility, seven-protein as a simple sample, high peak
conventional size exclusion chromatography capacity of 2D-plat f orm was achieved.
(SEC) has been widely used for the Innovative aspects
separation and purif ication of biomolecules.  Novel approach based on size
However, its existing shortcomings, such as excluson chromatography for high
poor resolution, low efficiency and narrow efficiency separation of proteins.
separation window, limited its application in  Construction of consecutive 2D-
proteome research. platform based on column switch
Recently, we developed a novel approac h Recycling size exclusion
for protein separation based on column chromatography.
switch recycling size exclusion  High peak capacity of 2D-platform
chromatography (CSRSEC), by which could be achieved.
proteins were alternatively switched from References
one SEC column to another, and then (1) Al-Somali, A. M.; Krueger, K. M.;
separated in terms of close-loop recycling. Falkner, J. C.; Colvin, V. L. Anal. Chem.
Such a system demonstrated improved 2004 , 76 , 5903-5910.
resolution for proteins and was further
applied for the construction of
multidimensional protein separation platform .
Methods
For CSRSEC analysis, pumps, SEC
columns, UV detector and two-position,
high-speed ten-port valves (Valco were Figure 1. Scheme of column switch
connected in terms of closed-loop (Figure1), recycling size exclusion chromatography
by which the late eluted components were (CSRSEC) platform.
first kept onto another column, and injected
after the early eluted ones were completely
separated. The CSRSEC approac h was
further integrated with RPLC to construct a
consecutive multidimensional platform for
proteome analysis.
Result s
By CSRSEC analysis, proteins with wide
molecule weight distribution were completely. Figure2. Separation of myoglobin and
The resolution of proteins with minor MW ribonuclease (2500Da difference) by
difference was greatly improved (Figure2). CSRSEC
Furthermore, the CSRSEC approac h was
 DEVELOPMENT OF MULTI-DIMENSIONAL CHROMATOGRAPHY-MASS
SPECTROMETRY TECHNIQUES FOR PROTEIN COMPLEX ANALYSIS

Protein-protein interaction is one of the challenge studies in functional proteome

research. Multi-dimensional chromatography (MDLC) and mass spectrometry
technologies are proved to be useful in protein-protein interaction studies. Some
non-denaturation chromatography, e.g. size exclusion chromatography (SEC), ion
exchange chromatography (IEC), and hydrophorbic interaction chromatography
(HIC), and various electrophoretic separations are readily used for separation and
purification of protein complexes in variety of biological samples. Meanwhile some
denatured chromatography and electrophoresis, reversed phase chromatography
(RPLC), and gel electrophoresis, SDS-PAGE, etc, are further used to separate the
complexes into single proteins. In this way, large amount of protein complexes can be
separated and identified and the possible protein-protein interactions can also be
found out in the analysis. In our work, two-dimensional chromatography, SEC-IEC,
was employed to separate hundreds of protein complexes. A denatured SDS-PAGE
were further run to compare the differences of protein compositions and verify the
molecular weights. Proteins from complexes were recognized and identified by mass
spectrometry and protein-labeled fluorescence for subjecting to further verifications.
Such a fast MDLC screening approach has great potential for finding large scale of
protein complexes existed in various biological systems. This approach could be used
to screening the protein-drug and even small molecules interactions to discover
molecules of biological significance. (This work is supported by 973 Project,
2007CB914100)
 A New Protein Equalizer Based on M13 scFv Displaying Library

Peng Zhao, Lihua Zhang, Zhen Liang, Yukui Zhang

National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of

Sciences, Dalian, China, 116023. lihuazhang@dicp.ac.cn

Introduction
The analysis of proteomes is a challenge, because Innovative aspects
the presence of high-abundance proteins can • Capability to deplete high-abundance proteins
overwhelm the signals of low abundant ones. Thus in unknown samples in which the specific
various methods for the depletion of high abundant antibodies of high-abundance proteins are not
proteins have been developed. Protein equalizer discovered.
technology is another means to reduce protein • Easy and low cost to prepared such sample
concentration differences, thus improving the preparation materials, suitable for the analysis
detection sensitivity of low abundance proteins. In of various proteome samples.
our recent study, a new protein equalizer based on
M13 scFv displaying library was developed, and
successfully applied to simultaneously deplete a References
number of high abundant proteins in human serum (1) Arvidsson. P et al, Direct chromatographic
and urine of nephropathy patients. capture of enzyme from crude
homogenate using immobilized metal
affinity chromatography on a continuous
Methods supermacroporous adsorbent. J.
The epoxy-containing supermacroporous Chromatogr. A, 2003, 986, 275–290.
monolithic cryogel was prepared according to (2) Kumar. A et al, Affinity fractionation of
reference (1). Then the amplified and purified lymphocytes using a monolithic cryogel. J.
whole M13 scFv displayed library was coupled to
cryogel by the “glutaraldehyde method” (2).
Immunol. Meth. 2003 ，283: 185–194.
Human serum and urine proteins of nephropathy
patient were selected to study the high abundance
protein depletion capacity of the new protein
equalizer. Each sample was added to the M13-
coupled cryogel for 60 min, then the eluants were
collected and analyzed by SDS-PAGE and MS.
Finally the cryogel was regenerated by 2M NaCl.

Results
Figure 1 showed the analysis of human serum and
urine proteins of nephropathy patients treated with
the protein equalizer by SDS-PAGE. It could be
seen that the concentration of proteins was
obviously equalized, by sharply reducing the
concentration of the most abundant components,
while simultaneously enhancing the concentration
of the low abundance species. In addition, the
MS/MS identification results also demonstrated
that after the treatment, more low concentration
proteins could be identified. Compared with affinity
depletion methods, the new protein equalizer with
M13 scFv displaying library immobilized is more
suitable for the depletion of various high-
abundance proteins in unknown complex samples.
At the same time, different low abundance proteins
could be enriched without bias. All these results
demonstrate that such a novel protein equalizer
might pave a new way for exploring more proteins
in proteome.
A B
Figure 1 .The results of depletion of high abundant
proteins using the new protein equalizer.
Samples were loaded onto each lane and separated by
12% gels. A: human serum proteins after depletion of
high abundant proteins. Lanes: 1, Marker (97, 66, 43,
31, 20 and 14kDa); 2, depleted serum using M13-
uncoupled cryogel; 3, depleted serum using M13-
coupled cryogel; 4, crude serum. B: urine proteins of
nephropathy patient after depletion of high abundant
proteins. Lanes: 1, Marker; 2, depleted urine proteins
using M13-uncoupled cryogel; 3, depleted urine proteins
using M13-coupled cryogel; 4, crude urine proteins of
nephropathy patient.