Biomarkers and omics

Compact Quantitative Proteomics Workflow Combining
SILAC Labeling, Chromatographic Pre-fractionation
and CESI-MS with a Neutral Capillary Surface• Fully automated reverse-phase chromatography fractionation and CESI-MS analyses• Increased coverage of modified peptides without sample enrichment• Reduced workload due to the single in-solution digestion• Ultralow sample consumption (c.a. 40nL) per analysis
Klaus Faserl,1 Leopold Kremser,1 Martin Müller,2 David Teis,2 László Hajba,3 András Guttman4 and Herbert H. Lindner1
1 Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria
2 Division of Cell Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria
3 MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary
4 SCIEX, Brea, CA, US

Overview

Introduction

Capillary electrophoresis coupled with mass spectrometry is

Quantitative proteomics recently has gained a high level ofa powerful combination of a high-performance liquid phaseinterest and is considered an essential tool in molecular biology

separation technique and a versatile detection method, providing

and biomedical sciences. This trend has been facilitated byexcellent selectivity, high sensitivity and structural information.

the rapid development of high-resolution mass spectrometers

CESI is the combination of electrospray ionization (ESI) withenabling fast and sensitive identification of proteins relevant

capillary electrophoresis (CE) in a single dynamic process. In

to biological processes. The basic workflow of quantitative

this work, an ultra-low flow CESI approach in combination withproteomics using a technique such as SILAC comprises thereversed-phase liquid chromatography pre-fractionation was

following steps: (i) stable isotope labeling of proteins or peptides;

applied for quantitative proteomics. Proteins were extracted from(ii) enzymatic digestion of these proteins into peptides; (iii)

SILAC (stable isotope labeling by amino acids in cell culture)

separation of peptides and (iv) mass spectrometry detection

labeled and unlabeled yeast strains, mixed and enzymaticallyand analysis. In most instances, a multi-dimensional separation

digested in solution. The resulting peptides were pre-fractionatedstrategy is included in the proteomic workflows because of theusing chromatography and the fractions were analyzed by CESI-

large number of proteins and cleaved peptides in the sample.

MS using a neutral surface capillary. A total of 28,538 peptides

The most commonly used techniques in the first dimension

were identified corresponding to 3,272 quantified proteins. CESI-

are sodium dodecyl sulfate-polyacrylamide gel electrophoresis

MS measurement was performed under ultra-low flow conditions(SDS-PAGE), off-gel isoelectric focusing (IEF) and LC-based(<10 nL/min) to obtain the highest separation efficiency withstrategies such as ion exchange, hydrophilic interaction (HILIC)

the neutral surface capillary. The CESI-MS approach appliedor reverse-phase chromatography.2-5 The latter is mostly used inalso proved to be a powerful method for identification of low-

acidic separation conditions as a second separation dimension.

abundance modified peptides within the same sample without

Benefits of these methods include MS compatibility with thethe need for further enrichment. Using the CESI-MS approach,solvents used for separation, and the ability to easily tune the1,371 phosphopeptides were successfully identified, 49 of which

separation conditions to deal with the complexity of the sample

were found to be differentially regulated in the 2 yeast strains.

and the scan speed of the MS instrument. However, this method

Apart from the 33,854 unique peptides found using this method,is less suitable for hydrophilic peptides, which can be lost during

8,106 acetylated, phosphorylated, deamidated or oxidized

the pre-column wash step, and phosphopeptides, which may

peptide forms were also identified. This technical note is based

undergo ion suppression due to co-eluting peptides.6,7 In order toon previously published results.1
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Biomarkers and omics
avoid these problems, efforts have been made to couple capillary

were compared to the corresponding wild type (WT) cells

electrophoresis (CE) with mass spectrometry to utilize the proven(vps4Δ, pRS413-VPS4).

high separation efficiency of CE for the separation of modified
proteins and peptides of any size.8-15

In-solution protein digestion: Cleared cell lysates (1.5
mg of extracted yeast proteins) were TCA-precipitated andOver the last decades, numerous interfaces have been designed

washed twice with acetone. The precipitated protein pellet wasand developed to enable efficient CE-MS coupling.

The

resuspended in ammonium bicarbonate (100 mM, pH 8.0).

sheath flow interface enables ESI voltage contact throughProteins were reduced with dithiothreitol (5 mM) at 56 °C fora constant flow of sheath liquid applied hydrodynamically

30 min and alkylated with iodoacetamide (18 mM) at room

or electrokinetically.18-20 A modified version of this interface

temperature for 20 min. Proteins were digested overnight at

is the “liquid junction.” Sheathless interfaces usually apply

37 °C by adding Lys-C at 1:75 ratio (protease/protein).

16-17

21

a steel needle at the terminus of the separation capillary toassure closure of the electric circuit for both the CE and the
ESI processes22 using conductive materials to coat the emittertip.23, 24 The flow rate toward the MS unit is influenced by theelectroosmotic flow and/or the applied pressure in the system.
CESI represents an advanced version of sheathless sprayers,utilizing a separation capillary with a porous tip acting as ananospray tip.25 The main advantage of using CESI is thecapability to operate at low nanoliter flow rates (<10 nL/min)
resulting in decreased ion suppression and overall improved
sensitivity.26-28 In analogous work, CESI-MS was successfully
applied for discovery of post-translational modifications on
antibodies and histones with high sensitivity.29-33
In this study, CESI-MS resulted in the highly sensitive quantitative
analysis of the yeast proteome. Extracted proteins from SILAC
labeled and unlabeled yeast strains were mixed and digested
enzymatically. Following digestion, the resulting peptides
were first fractionated with reverse-phase chromatography andReverse-phase chromatography fractionation: In-solution
digested peptides were loaded on a Beckman Gold HPLC
system (Beckman Coulter, Brea, CA) and fractionated byreverse-phase chromatography using an EC 250/4.6 Nucleosil
120-3 μm C18 column (Machery-Nagel, Düren, Germany).
Digested yeast proteins (1.4 mg) were eluted within 2 husing a constant flow rate of 0.5 mL/min. Eluents were 0.1%
trifluoroacetic acid (solvent A) and 0.1% trifluoroacetic acid in
85% acetonitrile (solvent B). The gradient started at 4% solvent
B for 14.5 min and increased to 60% solvent B in 90 min, up to
100% B in 4 min, and was held at 100% B for 11.5 min.
Fraction collection: Collection started 5 min after injection
at 0.5 min intervals for 80 min and 1 min intervals for another22 min. In total, 182 fractions were collected and then lyophilized
and stored dry at -20 °C. Prior to capillary electrophoresis,
the peptides were dissolved in 15 μL of 50 mM ammonium
acetate (pH 4.0).

then analyzed by CESI-MS using a neutral surface capillary

Capillary electrophoresis: The CESI 8000 Plus Highcolumn. CESI-MS data were analyzed to identify any post-

Performance Separation-ESI Module (SCIEX, Brea, CA) wastranslational modifications, primarily phosphorylated peptides

used with a 100 cm long 30 μm i.d. (150 μm o.d.) neutral surface(including phosphorylation sites) and other modifications

capillary with an integrated 3 cm long porous tip, serving bothsuch as acetylation, deamidation and oxidation.

as separation capillary and electrospray emitter. The CESI
capillary was coupled to an LTQ Orbitrap XL mass spectrometer

Materials and methods(Thermo Scientific, San Jose, CA) by inserting the porous

Chemicals: Dithiothreitol was purchased from Biomol (Hamburg,

to supply the sprayer housing with conductive liquid in order toGermany) and iodoacetamide from GE Healthcare (Vienna,
Austria). Yeast growth media was from Sunrise Science Products(CSM−His, −Arg, −Lys). 13C6 15N2-L-Lysine and Endoproteinase
Lys-C from Lysobacter enzymogenes and all other chemicals
were purchased from Sigma-Aldrich (Vienna, Austria).
Cell culture: MBY4 yeast strain was used (MATα leu2-3, 112
ura3-52 his3-200 trp1-901 lys2-801 suc2-9, vps4:TRP1)1 for all
experiments. Isogenic yeast ESCRT mutants (vps4Δ, pRS413

segment into the sprayer interface.1 A second capillary was usedprovide electrical contact. 10% (v/v) acetic acid was used bothas background electrolyte (BGE) and conductive liquid for theemitter. Prior to capillary electrophoresis, both the separationand the conductive liquid capillaries were rinsed with fresh
buffer. The sample was introduced by applying 5 psi pressurefor 50 s (40 nL injection volume), followed by a plug of BGE(5 psi for 5 s). The applied electric voltage was +30 kV with asimultaneous pressure of 1 psi for 60 min at the capillary inlet
resulting in an approximate flow rate of 10 nL/min.
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Biomarkers and omics
Important:corresponding to ~6% of the total column volume. Please• A separation current above 5 μA might cause permanent
damage to the separation capillary.

2 μL aliquots, 325 injections could be performed with 40 nL• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

note that re-dissolving the LC fractions in 15 μL and using
injection volumes.

CESI−MS analysis of the yeast proteome

Mass spectrometry: The mass spectrometer (LTQ Orbitrap XL,

CESI-MS analysis of all 182 fractions with subsequent database

Thermo Scientific) was used in data-dependent mode to switch

search using the Proteome Discoverer software was performed

between MS and MS/MS acquisition. Survey full scan MS spectra

and resulted in 33,656 identified peptides (modified forms

were acquired in the Orbitrap with a resolution of R = 60,000

not included). Of these peptides, 28,536 were quantified,(at m/z = 400) in profile mode after accumulation to an automatedcorresponding to ~85% quantification rate. The remaining

gain control (AGC) target value of 1 × 106 in the linear ion

non-quantified 5,120 peptides included: 2,254 (44%) peptides

trap. MS/MS spectra were obtained in the linear ion trap (LTQ)with no lysine in their sequence (837 C-terminal and 1,417

using collision induced dissociation (CID). The 6 most intense

non-specifically cleaved peptides); 1,124 (22%) peptides withprecursors were sequentially selected for MS/MS fragmentation.

non-unique protein sequence and 1,742 (34%) peptides, which

Parameters applied for fragmentation were as follows: minimum

were not quantified due to low signal intensity or overlapping

signal required 1000; isolation width (m/z) 2.0; activation time

peptide isotopic distributions.

30 ms; normalized collision energy 35.0 and activation Q of
0.250. MS/MS spectra were acquired in centroid mode with an
AGC target value of 1 × 104 and 100 ms maximum ionization
time, respectively. Dynamic exclusion was set to 15 s.

The largest number of peptides was found in fractions 55–150
and approximately 86% of all quantified peptides were in thesefractions. Hydrophilic peptides contributed approximately 7%(fractions 1–54), and very hydrophobic peptides accounted for

Data analysis and quantification: Proteome Discoverer version

approximately 8% (> fraction No 150). In addition, 3,429 proteins

1.4.0.288 (Thermo Scientific) and MaxQuant version 1.3.0.5 were

were identified with at least 2 unique peptides and 3,272 proteinsused for data analysis. Raw data obtained by CESI-MS were

were quantified with at least 2 unique peptides and 2 peptide

searched against a yeast ORF database downloaded from theH/L ratios.

SGD Saccharomyces Genome Database (www.yeastgenome.
org; 6,627 entries, last modified February 3, 2011).

Results
The aim of this study was to test the application of ultra-low
flow capillary electrophoresis mass spectrometry couplingfor SILAC-based quantitative proteomics with a special focus
on specific post-translational modifications. Protein extracts of
2 isogenic yeast strains (a heavy-lysine labeled wild-type and anon-labeled mutant) were mixed in a 1:1 ratio and enzymaticallydigested by Lys-C in solution. The resulting peptides were thenfractionated by reverse-phase chromatography. To circumvent
losing the hydrophilic peptides, which otherwise poorly interact
with the reverse-phase material, the separation was initiatedin low organic isocratic mode (3% acetonitrile) and continued
by gradient elution up to 85% acetonitrile with a total separation
time of 120 min. Highest UV absorbance was observed between
55 and 70 min, suggesting that these fractions contained thegreatest number of peptides. The collected 182 fractions werethen analyzed with CESI-MS employing a neutral surface

High efficiency was observed during the RP chromatographic
separation of early and middle eluting peptide fractions,while slightly reduced separation efficiency was obtained forhydrophobic peptides in the later fractions. In total, 55% of all
peptides were quantified in a single fraction and 29% in 2 otherreactions, which indicated excellent separation efficiency. Only
5% of peptides, mainly hydrophobic, were quantified in morethan 5 fractions. The total time required to analyze all 182
fractions was 215 h (the MS data acquisition time was 182 h).
The proteins identified with absolute cellular protein abundanceswere compared to literature data,35 and the CESI-MS approach
identified nearly all high-abundance proteins (>104 copies percell), as shown in Figure 1. Also, a large number of mediumand low-abundance proteins (<104 copies per cell) wereidentified. Please note that CESI-MS was even able toidentify very low-abundance proteins. These results clearly
indicate that the CESI-MS approach is quite powerful for highsensitivity protein identification. These results also highlight themultidimensional fashion of chromatographic pre-fractionation
and CESI-MS analysis.

capillary column. 40 nL sample was introduced into the capillary,p3

Biomarkers and omics

Figure 1. The ability of CESI-MS to identify proteins at various
abundance levels.

Figure 2. Quantification of phosphopeptides by CESI-MS.

Analysis of phosphopeptides

Analysis of other post translational modifications

Additional database searches of the obtained mass spectra

The CESI-MS data sets were also searched for the presence ofwere performed using 3 different search engines (Sequest,

PTMs such as acetylated, deamidated and oxidized peptides.

Mascot and Andromeda) to identify phosphorylation levels.

This additional database search revealed the existence of 6,623

As a result, 1,483 phosphorylated peptides, identified bymodified peptides (Figure 4). A large number of peptides wasat least 2 of the search programs, were selected for further

found containing 1 (3,860) and 2 (403) deamidated asparagines.

investigations. As depicted in Figure 2, detailed data analysis

Moreover, 900 proteins were found to be co-translationally andrevealed the presence of 1,274 mono-, 195 di-, 12 tri- and153 peptides post-translationally acetylated on N-terminal and2 tetra-phosphorylated peptides with a total of 1,371 peptides

lysine residues, respectively. Please note that acetylation ofquantified. 1,127 modification sites were assigned with >95%

these amino groups lowers the net charge of these peptides

accuracy according to localization scores calculated with

causing lower electrophoretic mobility. Consequently, most ofProteome Discoverer and MaxQuant software. The number

these acetylated peptides appeared at the higher migration timeof phosphopeptides identified by CESI-MS was rather high,range of >30 min, similar to that of phosphopeptides, as shown

especially bearing in mind that no enrichment strategy was used.

in Figure 5. The separation of acetylated and phosphorylatedThis phenomenon can be explained by the greatly reduced ion

peptides from their non-modified counterparts at a region where

suppression inherent to CESI at very low flow rate conditions

only a few solute molecules migrate enables high sensitivityof 10 nL/min. On the other hand, phosphopeptides migrated

identification of even very low abundant peptides.

significantly slower than most of the regular peptides present
in the fractions because of their reduced net charge at the
pH of the background electrolyte.
Light/heavy (L/H) ratios of phosphopeptides, the proteins
corresponding to them and the change in phosphorylation levelare depicted in Figure 3. According to the results, 50 peptides
were found to be variably abundant in heavy and light labeled
yeast strains, and 16 phosphopeptides were not significantly
regulated when their expression level was corrected by thecorresponding protein expression. On the other hand, an
additional set of 15 peptides became significantly regulated
for the same reason.
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Biomarkers and omics

Figure 3. Light/heavy (L/H) ratios of phosphopeptides, the proteins corresponding to them and the change in phosphorylation level.

Figure 5. CESI-MS separation of peptides also showing the intensity
and the particular time of quantification in chromatography fraction.
Black bars, 688 non-modified peptides; yellow bars, 22 acetylated
peptides; blue bars, 57 phosphorylated peptides.

Figure 4. Modified peptides (except phosphopeptides) found in the
CESI-MS data set of the 182 LC fractions analyzed.

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Biomarkers and omics
Conclusions

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analysis for relative quantification of SILAC labeled yeast strains
has been presented. A very large number of phosphopeptides
and other modified peptides (e.g., acetylated, deaminated, etc.)
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4. Because of the small sample consumption of CESI-MS(c.a. 40 nL), the samples can be easily reanalyzed orstored for later use.

Acknowledgement
This work was funded in part by the Austrian Science Fund(FWF), Grant Y444-B12 and the joint research project (AT-HU).

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