Technical Note

pubs.acs.org/jpr

Rapid Combinatorial ERLICSCX Solid-Phase Extraction for In-Depth

Phosphoproteome Analysis
Mostafa Zarei,*,,, Adrian Sprenger,,,, Christine Gretzmeier,, and Joern Dengjel*,,,,

Freiburg Institute for Advanced Studies (FRIAS), School of Life Sciences-LifeNet, University of Freiburg, Albertstr. 19, 79104
Freiburg, Germany

ZBSA Center for Biological Systems Analysis, University of Freiburg, Habsburgerstr. 49, 79104 Freiburg, Germany

Department of Dermatology, University Freiburg Medical Center, Hauptstr. 7, 79104 Freiburg, Germany

BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schanzlestr. 18, 79104 Freiburg, Germany
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Publication Date (Web): November 4, 2013 | doi: 10.1021/pr4007969

S Supporting Information
*

ABSTRACT: Protein phosphorylation is an important mechanism

of cellular signaling, and many proteins are precisely regulated
through the interplay of stimulatory and inhibitory phosphorylation
sites. Phosphoproteomics oers great opportunities to unravel this
complex interplay, generating a mechanistic understanding of vital
cellular processes. However, protein phosphorylation is substoichiometric and, in particular, peptides carrying multiple phosphorylation
sites are extremely dicult to detect in a highly complex mixture of
abundant nonphosphorylated peptides. Chromatographic methods
are employed to reduce sample complexity and thereby signicantly
increase the number of phosphopeptide identications. We
previously demonstrated that combinatorial strong cation exchangeelectrostatic repulsionhydrophilic interaction chromatography yields a surplus in overall identications of phosphopeptides compared with single chromatographic approaches. Here we
present a simple and rapid strategy implemented as solid-phase extraction not requiring specic instrumentation such as o-line
HPLC systems. It is inexpensive, adaptable for high and low amounts of starting material, and saves time by allowing multiplexed
sample preparation without any carry-over problem.
KEYWORDS: SPE, ERLIC, SCX, phosphoproteomics, TiO2, chromatography, mass spectrometry

INTRODUCTION
Protein phosphorylation represents one of the most important
mechanisms of cellular signal transduction,1 inuencing among
others protein stability, activity, subcellular localization, and
anity.2 Phosphorylation introduces a polar charge that can
induce a conformational change in proteins or can block or free
binding sites. Frequently, proteins are regulated through the
concerted action of several phosphorylation sites.3 Mass
spectrometry (MS) enables unbiased large-scale analyses of
the proteome and its post-translational modication.4 Prior to
MS analysis, proteins are commonly digested, as peptides can
be more easily separated and ionized. Phosphorylation sites are
recognized through a mass shift of 79.9963 Da5 and can be
specically addressed in mass spectrometric analysis strategies,
such as multistage activation (MSA),6 higher energy collisional
dissociation (HCD), or collision-induced dissociation (CID).7
If the MS/MS spectrum is of certain quality, a unique
localization of the phosphorylation site within a peptide is
possible.8
However, in-depth proteome analysis is hampered by the
enormous complexity of whole cell lysate digests and the high
dynamic range of protein abundances. The analysis of the
2013 American Chemical Society

phosphoproteome adds additional challenges. Because potential

phosphorylation sites are generally occupied at substoichiometric levels, phosphopeptides are low abundant and in a
complex mixture of excess nonphosphorylated peptides. Only
with phospho-anity purication methods, such as immobilized metal ion chromatography (IMAC)9 and TiO2 anity
chromatography,10,11 phosphopeptides can be enriched to an
extent that enables ecient MS-based phosphoproteome
analysis. 30% of all proteins are believed to carry at least one
phosphorylation site.4,12 The many possible occupation
combinations of peptides with multiple phosphylation sites
tremendously increase the complexity of the sample. Rapid
technological advances in mass spectrometers account for this
by increasing scanning speed and sensitivity.1315 However,
biological sample complexity still exceeds the separation power
and acquisition speed of state-of-the-art LCMS combinations.16 Thus, for in-depth analyses, fractionation and enrichment of phosphopeptides prior to LCMS/MS analysis is still
state-of-the-art.17,18 Dierent physicochemical traits of phosReceived: August 1, 2013
Published: October 22, 2013
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Technical Note

Figure 1. Experimental workow. Proteins in whole cell lysate were digested using trypsin. Peptides were fractionated by ERLICSPE and SCX
SPE, respectively. The FT from the ERLIC column was loaded onto the SCXSPE column. In total, ve fractions were collected from each step,
plus the FT from SCX. All 10 fractions were subjected to one-time TiO2-based phosphopeptide enrichment, the FT to three consecutive times,
yielding 13 phosphopeptide samples that were analyzed by RPLCMS/MS.

FPLC generally requires large sample amounts. To address

these drawbacks, we transfer the principle of combinatorial
phosphopeptide fractionation by ERLICSCX to a self-packed
solid-phase extraction (SPE) setup and demonstrate high
phosphopeptide separation and identication rates for single as
well as multiple phosphorylated peptides. The SPE procedure is
inexpensive and easy-to-use, enabling a quick implementation
in any laboratory.28 Moreover, it allows parallel handling of
ERLIC and SCX samples saving a substantial amount of time
compared with traditional FPLC applications.

phopeptides can be addressed for optimal fractionation, for

example, hydrophobicity, hydrophilicity, or charge.17,19 The
combinatorial use of two chromatographic methods addressing
inverse or complementary physicochemical characteristics of
phosphopeptides consequently yields an improved fractionation. Oine SCX fractionation coupled to online RPRP
(high- and low-pH), RPTiO2RP, or SCXWAX all have
been described in recent studies.2023
We recently proposed the combinatorial use of ERLIC and
SCX to achieve ecient retention and fractionation, especially
of very low abundant multiple phosphorylated peptides.24 Both
methods are performed at low pH (<2.7) to uncharge acidic
peptides and retain the negative charges of phosphate groups.
Under these conditions, most tryptic peptides have a solution
charge state of +2, decreasing by 1 for each attached
phosphate group.12,25 Hence, the positively charged stationary
phase of ERLIC favors retention and separation of negatively
charged multiphosphorylated peptides, while the negatively
charged stationary phase of SCX favors retention and
separation of unphosphorylated or singly phosphorylated
peptides.26,27 In this way, the very low abundant multiple
phosphorylated peptides are separated from the more abundant
singly phosphorylated peptides, increasing their identication
rate immensely.
A routine application of this method in average life science
laboratories is hampered by several factors: (1) Expertise in the
use of FPLC systems in dierent chromatographic modes is
required. (2) Switching between dierent chromatographic
modes is laborious and time-consuming;. (3) The use of a

MATERIAL AND METHODS

Chemicals

All solvents, acetonitrile (ACN) (Wako, Neuss, Germany),

acetic acid (LGC Promochem, Wesel, Germany), formic acid
(FA), and triuoroacetic acid (TFA) (Merck, Darmstadt,
Germany), were LC-MS grade. Sodium deoxycholate, dithiothreitol (DTT), ammonium bicarbonate (ABC), iodoacetamide
(IAA), lactic acid, triethylamine (TEA), sodium dihydrogen
phosphate (NaH2PO4), and sodium acetate were all purchased
from Sigma Aldrich (Munich, Germany). Potassium dihydrogen phosphate (KH2PO4) and potassium chloride (KCl) were
purchased from Merck. Methylphosphonic acid (MePO4) was
from Alfa Aesar (Karlsruhe, Germany). Sequencing grade
trypsin was purchased from Promega (Mannheim, Germany).
Water used in the experiments was prepared using a Milli-Q
system (Millipore, Bedford, MA).
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Sample Preparation

fraction and FT and incubated for 30 min at room

temperature.31 Beads were washed with 100 L of 10% ACN
and 1% TFA, followed by 100 L of 80% ACN and 1% TFA
and nally 100 L of water. Phosphopeptides were eluted using
25% ammonium hydroxide in 20 and 40% ACN, respectively.
Eluted phosphopeptides were dried to <5 L and resuspended
in 15 L of 0.5% acetic acid in water for analysis. Five L of
each fraction was used for MS analysis. For consecutive
incubations, the peptide-beads slurry was spun down, and the
supernatant was incubated with another aliquot of freshly
prepared TiO2 beads. Average enrichment of TiO2 on dierent
chromatography systems was calculated as the number of
detected unique phosphopeptides divided by the total number
of detected unique peptides per fraction.

HeLa cells were grown to near conuence in 15 cm dishes and

scraped and lysed in 1% sodium deoxycholate in 50 mM ABC
buer, and the lysate was claried by centrifugation.29,30
Supernatants were reduced with 1 mM DTT and alkylated
with 5.5 mM IAA for 45 min at room temperature in darkness
before being digested with trypsin (1:75 trypsin/protein)
overnight at 37 C. Digests were acidied to 1% FA, and
undigested proteins and deoxycholate were removed by
centrifugation. The protein concentration was measured using
the bicinchoninic acid assay (Thermo Fisher Scientic, Bonn,
Germany), and tryptic digests were stored at 80 C for further
use.

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Publication Date (Web): November 4, 2013 | doi: 10.1021/pr4007969

Preparation of ERLIC and SCXSPE Material

Mass Spectrometric Analysis

For fractionation by ERLIC and SCXSPE, respective bulk

material was self-packed into 1 mL pipet tips with punched
Whatman paper as frit in the front part (Figure 1). One g of
solid-phase slurry of each ERLIC Poly-WAX LP (5 m particle
size, 300 pore size; PolyLC, Columbia, USA) and SCX
PolySULFOETHYL A (5 m particle size, 300 pore size;
PolyLC), was prepared according to the following protocol: (1)
Transfer both solid-phase materials to separate 50 mL reaction
tubes and wet beads using 25 mL of methanol by soft rotation
at room temperature (RT) for 10 min. (2) Centrifuge tubes at
0.2 rcf and wash beads with 5 mL of water. (3) Wet beads with
40 mL of 0.2 M sodium dihydrogen phosphate (NaH2PO4) and
0.3 M sodium acetate in water for 1 h at RT by soft mixing. (4)
Wash beads with 5 mL of water and add water to the solid
phase to reach the nal volume of 4 mL. For each SPE
experiment, we used 1 mL of these slurries that are
representative of 250 mg of respective starting materials. (5)
Transfer 1 mL of solid phase slurry to the blocked pipet tips
and ush it with 1 mL of respective solvent B and (6) by a
minimum of 5 mL of respective solvent A. (7) Load the sample
to the beads and elute with dierent ratios of solvent B/A (see
workow section). In all steps, the liquid was eluted by
centrifugation at 0.8 rcf. Two cm of the top part of pipet tips
was cut to t them into a tabletop centrifuge.

Mass spectrometric measurements were performed on an LTQ

Orbitrap XL mass spectrometer (Thermo Fisher Scientic,
Bremen, Germany) coupled to an Agilent 1200 (Agilent
Technologies, Waldbronn, Germany) or an Eksigent 2D
nanoow-HPLC (AB Sciex, Darmstadt, Germany). HPLCcolumn tips (fused silica) with 75 m inner diameter (New
Objective, Woburn, MA) were self-packed with Reprosil-Pur
120 ODS-3 (Dr. Maisch, Ammerbuch, Germany) to a length of
20 cm. Samples were applied directly onto the column without
precolumn. A gradient of A [0.5% acetic acid in water] and B
[0.5% acetic acid in 80% ACN/water] with increasing organic
proportion was used for peptide separation (loading of sample
with 2% of solvent B; rst separation ramp from 2 to 35% B
within 100 min, and second ramp from 35 to 80% B within 20
min). The ow rate was 250 nL/min and for sample application
500 nL/min. Two technical replicates with identical settings
were performed. The mass spectrometer was operated in the
data-dependent mode and switched automatically between MS
(max. of 1 106 ions) and MS/MS. Each MS scan was
followed by a maximum of ve MS/MS scans in the linear ion
trap using 35% collision energy and a target value of 5000.
Parent ions with a charge state of z = 1 and unassigned charge
states were excluded for fragmentation. Further MS/MS
settings were: repeat duration (30 s), repeat count (1),
exclusion duration (45 s), and isolation width (2). For MS/
MS, wideband activation and multi stage activation were
enabled with the neutral loss mass list of singly, doubly, and
triply phosphorylated peptides. Remaining settings were set to
default. The mass range for MS was m/z = 350 to 2000, and
signal threshold was 1000. The resolution was set to 60 000.
Mass-spectrometric parameters were as follows: spray voltage
2.3 kV; no sheath and auxiliary gas ow; ion-transfer tube
temperature 200 C.

Workow of ERLICSCXSPE

Six mg of HeLa cell tryptic protein digest was acidied by FA to

a pH under 3, diluted to 1 mL with solvent A of ERLIC, and
loaded a minimum of two times to an equilibrated ERLICSPE
tip. The ow through (FT) was collected, concentrated to
reduce the ACN concentration to 30%, acidied with FA, and
loaded a minimum of two times to an equilibrated SCXSPE
column. Afterward, peptides on both SPE columns were
fractionated in ve steps by 1 mL of solvent B/solvent A in
dierent ratios. For ERLIC, 20, 40, 60, 80, and 100% of solvent
B were chosen, while for SCX, 10, 20, 30, 40 and 100% solvent
B provided greater fractionation eciency. These percentages
were determined in setup experiments (data not shown).
For ERLIC, 10 mM sodium methylphosphonate (NaMePO4) with 70% ACN, pH 2.0, and 200 mM triethylamine
phosphate (TEAP) with 60% ACN, pH 2.0 were used as
solvent A and solvent B, respectively. In SCX, 30% ACN
containing 5 mM KH2PO4, pH 2.7, was used as solvent A and
30% ACN containing 5 mM KH2PO4 and 300 mM KCl, pH
2.7, was used as solvent B.

Data Analysis

All raw les were analyzed with the software MaxQuant

(version 1.0.1.18).32,33 Cysteine carbamidomethylation was
selected as xed modication; methionine oxidation, protein
N-terminal acetylation, and phosphorylation on serine,
threonine, and tyrosine were selected as variable modications.
Up to three miscleavages were allowed. Precursor ion mass
tolerance was 6 ppm, and fragment ion mass tolerance was 0.5
Da for MS/MS spectra. A false discovery rate (FDR) of 1% and
a minimum length of six amino acids were used for
phosphopeptide and site identications. The Evidence table
was basis for peptide analyses. Two peptides identied by two
sequence spectra were regarded as unique if they (a) diered in
their amino acid sequence or (b) diered in their

TiO2 Enrichment

Ten L of a 30% (V/V) TiO2 slurry (MZ-Analysentechnik,

Mainz, Germany) in 30 mg/mL lactic acid was added to each
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Technical Note

phosphorylation site(s). Dierent charge states were not taken

into account.

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Publication Date (Web): November 4, 2013 | doi: 10.1021/pr4007969

RESULTS AND DISCUSSION

For the combinatorial ERLICSCXSPE workow for
phosphopeptide fractionation, HeLa cell lysate was used
(Figure 1). Six mg cells were lysed using sodium deoxycholate,
resulting in 1 mL of cell lysate and digested with trypsin.
Peptides were taken up in ERLIC buer A and loaded onto the
ERLICSPE column. (See the Material and Methods for
details.) The ERLIC FT was concentrated in a SpeedVac to
reduce the concentration of acetonitrile to 30%, acidied by FA,
loaded onto the SCXSPE column, and the SCX FT was again
collected. In contrast with an HPLC-based workow, it should
be considered that in the SPE approach the columns are packed
only by centrifugation and not by high pressure. The resulting
dead volume between the beads is rather high and results in less
ecient peptide binding. Therefore, the samples were reloaded
up to three times onto the same tip to improve binding.
Peptides from ERLICSPE and SCXSPE columns were each
eluted in ve steps using increasing percentages of respective
elution buers. (See the Material and Methods for details.)
Each fraction was subjected to one TiO2-based phosphopeptide
enrichment step, except the SCX FT, which was incubated
three times. The resulting 13 phosphopeptide samples were
analyzed by LCMS/MS using collision-induced dissociation
and multistage activation as fragmentation technique. Thirteen
145 min runs gave rise to a total MS measurement time of 32
h per experiment. MaxQuant and Andromeda were employed
for peptide identication.33 All experiments were performed
minimally in two technical replicates, starting from the
ERLICSPE step. (See Supplementary Figure S1 and Table
S1 in the Supporting Information.)
In the combinatorial workow, 13 fractions yielded a total of
51 244 peptidespectrum matches (PSMs), of which 42 012
could be matched to phosphopeptides. 9952 of these
phosphopeptides were unique, and of these 2137 were multiply
phosphorylated (3p). The peptides gave rise to more than
5670 phosphorylation sites on 2198 proteins. The complete list
of identied unique phosphopeptides is available as Supplementary Table S2 in the Supporting Information. Phosphopeptide enrichment eciency was on average 93% for the ERLIC
fractions and 71% for the SCX fractions. When measurement
time is scarce, SCX fractions 4 and 5 can be omitted with only
7% data loss (Figure 2A). We recommend not to omit any
fractions of the ERLIC approach because more than 80% of the
unique phosphopeptides found in fractions 4 and 5 are usually
overlooked, as they are low-abundant, carrying multiple
phosphate groups.
To conrm separation according to the inherent anity of
negative charges in ERLIC and positive charges in SCX,
respectively, we analyzed the average solution net charge of
identied peptides in each fraction. In the ERLIC fractions, we
observe the expected decrease in average peptide net charge
with increasing fraction number; in the SCX fractions, we
observe the expected increase (Figure 2B). An increase in
charge state in the consecutive FT phosphopeptide enrichment
steps can also be observed, as multiphosphorylated peptides
have a stronger anity to TiO2 and are consequently depleted
rst (data not shown). Of all phosphopeptides identied in all
ERLIC and SCX fractions, 69% are found in just one fraction,
and 20% are shared between two fractions, indicating a fair
fractionation quality throughout the two dierent chromato-

Figure 2. Yield of the combinatorial ERLICSCXSPE workow.

(A) Number of unique phosphopeptides (green bars), unique
unphosphorylated peptides (gray bars) in each fraction, and
cumulative count (red line) are shown. pp = phosphopeptide. (B)
Average solution net charge state of peptides in respective fractions,
(C) overall peptide overlap between fractions as the percentage of
peptides that are found in 1, 2, 3, or more fractions, and (D) detailed
overview of peptide overlap, showing how peptides assigned to a
specic fraction (by median-normalized intensity) distribute across all
other fractions.

graphic methods (Figure 2C). To get a more detailed overview

of fraction overlap between both chromatographic methods, we
visualize the overlap of each fraction with all other fractions.
For this purpose, we assigned phosphopeptides primarily to
that fraction in which they were found to be most abundant
based on median-normalized intensities (Figure 2D). We found
the largest overlap between the FT and the rst fractions of
ERLIC and SCX, respectively. This can be expected because
these fractions select for similar solution charge states (Figure
2B). Also, peptides eluting in the very rst fraction of any
chromatographic separation generally have the lowest binding
anity to the solid phase and consequently can also be found
to a large portion in the respective FT.
ERLIC and SCX are complementary, as indicated by the
small overlap of unique phosphopeptides between the two
consecutive separation steps. 3757 (38%) unique phosphopeptides were found in the ERLIC fractions, 4653 (47%) were
found in the SCX fractions, and only 1542 (15%) were found in
fractions of both methods (Supplementary Table S2 in the
Supporting Information) (Figure 3A). The orthogonality of the
two approaches is also highlighted by their ability to separate
multiply and singly phosphorylated peptides, respectively
(Figure 3B). In ERLIC, the fraction size of multiphosphorylated peptides (3p) is on average 31% and goes up to 92% in
fraction ve. In contrast with that, SCX retains maximally 11%
multiphosphorylated peptides. ERLIC fractions 24 are the
most interesting in this regard, contributing each on average
336 unique multiphosphorylated peptides to the whole data set.
Fraction 5 consists of 14% of peptides carrying more than four
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Figure 3. Complementarity of ERLIC and SCX: (A) Overlap of unique phosphopeptides between ERLIC and SCX. (B) Percentage of nonunique
peptides with dierent degree of phosphorylation in each single fraction. (C) Number of unique multiphosphorylated peptides identied in dierent
ERLIC fractions plus cumulative count (pp = phosphopeptide). (D) Percentage of unique peptides with dierent degree of phosphorylation in
ERLIC fractions, SCX fractions + FT, and in the combined workow. (E) Pie chart illustrating the contribution of ERLIC and SCX to the
identication of singly/doubly and multiple phosphorylated unique peptides.

phosphorylation sites (Figure 3C). ERLIC fractions contained

in total 1685 (32%) unique multiphosphorylated peptides,
while SCX fractions contained only 576 (9%) unique
multiphosphorylated peptides. The combined ERLICSCX
fractionation yields a total of 2137 (22%) unique multiphosphorylated peptides (Figure 3D). Of these, 79% are
contributed by the ERLIC part and only 21% by the SCX part.
Of all singly/doubly phosphorylated unique peptides, the
majority is identied in the SCX fractions (60%; Figure 3E).
To compare the combinatorial fractionation using SPE with a
respective FPLC-based approach, we used data from the
current study and from our already published work.24 In both
cases, we used the same amount of sample and the same
instrumental setup. Both approaches yielded very similar results
with an identication of 386 phosphopeptides per hour in case
of the FPLC approach versus 377 phosphopeptides per hour in
the case of the SPE approach. The dierences in total numbers
of identied phosphopeptides (6433 versus 9552) can be
explained by the dierent LC gradient lengths. However, with
22% unique multiple phosphorylated peptides, we achieved a
three-fold increase in the SPE workow compared with the
FPLC workow. One reason for this might be the ve times
smaller amount of stationary phase used in the SPE workow.
The sample amount of 6 mg likely exhausted the binding
capacity of the ERLIC stationary phase primarily capturing the
highly ane multiphosphorylated peptides. Lower abundance
of singly phosphorylated peptides in the respective fractions
increases MS fragmentation frequency of multiphosphorylated
peptides, and hence identication rates. This demonstrates that
adaptation of the amount of stationary phase to the amount of
sample is an important factor to ne-tune the complementary
use of ERLICSCXSPE. The MaxQuant analysis of the
ERLIC fractions identied with 30% peptide-spectrum matches
relatively few peptides from the recorded MS/MS spectra. This

suggests that the choice of the MS fragmentation technique is

another opportunity for ne-tuning. Although more than 31%
of the detected phosphopeptides in the ERLIC fractions were
multiphosphorylated, we think this number could be even
further increased by use of ETD or HCD as a complementary
fragmentation technique to CID.34,35
A major advantage of the SPE-based workow is its
suitability for large as well as small amounts of starting
material. In contrast with the tubing in a FPLC-based workow,
the SPE setup presents only little hydrophobic surfaces that
potentially adsorb peptides, and hence it requires less starting
material. To demonstrate this, we performed a 1D SPE using
SCX only. We loaded 500 g of HeLa cell lysate onto 200 mg
of the respective stationary phase packed in a 1 mL pipet tip.
Five fractions plus FT were collected, phosphopeptides were
enriched using TiO2, and samples were analyzed by LCMS/
MS. We identied 1044 unique phosphopeptides (Supplementary Table S3 in the Supporting Information), which is a
fair amount in our instrumental setup.

CONCLUSIONS
Considering the importance of protein phosphorylation in
physiological and pathological signal transduction, phosphoproteome analysis holds great value in understanding the
underlying molecular mechanisms leading cellular phenotypes.
The combinatorial ERLICSCXSPE setup described here
makes sample preparation for in-depth phosphoproteome
analysis accessible for a broad range of biological laboratories
without the need for detailed expertise in chromatographic
methods and access to respective equipment. It is inexpensive,
suitable for limited samples amount, for example, from primary
cells, and saves time and labor by allowing multiplexed sample
preparation. With 22% of all identied phosphopeptides being
multiple phosphorylated (3p), the method enables the large5993

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(10) Kuroda, I.; Shintani, Y.; Motokawa, M.; Abe, S.; Furuno, M.
Phosphopeptide-selective column-switching RP-HPLC with a titania
precolumn. Anal. Sci. 2004, 20, 13131319.
(11) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.;
Jrgensen, T. J. D. Highly selective enrichment of phosphorylated
peptides from peptide mixtures using titanium dioxide microcolumns.
Mol. Cell Proteomics 2005, 4, 873886.
(12) Beausoleil, S. A.; et al. Large-scale characterization of HeLa cell
nuclear phosphoproteins. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
1213012135.
(13) Kocher, T.; Swart, R.; Mechtler, K. Ultra-high-pressure RPLC
hyphenated to an LTQ-Orbitrap Velos reveals a linear relation
between peak capacity and number of identified peptides. Anal. Chem.
2011, 83, 26992704.
(14) Michalski, A.; et al. Mass spectrometry-based proteomics using
Q Exactive, a high-performance benchtop quadrupole Orbitrap mass
spectrometer. Mol. Cell Proteomics 2011, 10, M111.011015.
(15) Oppermann, F. S.; et al. Comparison of SILAC and mTRAQ
quantification for phosphoproteomics on a quadrupole orbitrap mass
spectrometer. J. Proteome Res. 2013, 12, 40894100.
(16) Michalski, A.; Cox, J.; Mann, M. More than 100,000 detectable
peptide species elute in single shotgun proteomics runs but the
majority is inaccessible to data-dependent LC-MS/MS. J. Proteome Res.
2011, 10, 17851793.
(17) Engholm-Keller, K.; Larsen, M. R. Technologies and challenges
in large-scale phosphoproteomics. Proteomics 2013, 13, 910931.
(18) Rigbolt, K. T. G.; Blagoev, B. Quantitative phosphoproteomics
to characterize signaling networks. Semin. Cell Dev. Biol. 2012, 23,
863871.
(19) McNulty, D. E.; Annan, R. S. Hydrophilic interaction
chromatography reduces the complexity of the phosphoproteome
and improves global phosphopeptide isolation and detection. Mol. Cell
Proteomics 2008, 7, 971980.
(20) Hennrich, M. L.; Groenewold, V.; Kops, G. J. P. L.; Heck, A. J.
R.; Mohammed, S. Improving depth in phosphoproteomics by using a
strong cation exchange-weak anion exchange-reversed phase multidimensional separation approach. Anal. Chem. 2011, 83, 71377143.
(21) Lemeer, S.; et al. Online automated in vivo zebrafish
phosphoproteomics: from large-scale analysis down to a single
embryo. J. Proteome Res. 2008, 7, 15551564.
(22) Song, C.; et al. Reversed-phase-reversed-phase liquid
chromatography approach with high orthogonality for multidimensional separation of phosphopeptides. Anal. Chem. 2010, 82, 5356.
(23) Zhang, X.; et al. Multi-dimensional liquid chromatography in
proteomicsa review. Anal. Chim. Acta 2010, 664, 101113.
(24) Zarei, M.; Sprenger, A.; Gretzmeier, C.; Dengjel, J.
Combinatorial use of electrostatic repulsion-hydrophilic interaction
chromatography (ERLIC) and strong cation exchange (SCX)
chromatography for in-depth phosphoproteome analysis. J. Proteome
Res. 2012, 11, 42694276.
(25) Villen, J.; et al. Large-scale phosphorylation analysis of mouse
liver. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14881493.
(26) Alpert, A. J. Electrostatic repulsion hydrophilic interaction
chromatography for isocratic separation of charged solutes and
selective isolation of phosphopeptides. Anal. Chem. 2008, 80, 6276.
(27) Zarei, M.; Sprenger, A.; Metzger, F.; Gretzmeier, C.; Dengjel, J.
Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global
phosphoproteomics approaches. J. Proteome Res. 2011, 10, 3474
3483.
(28) Dephoure, N.; Gygi, S. P. A solid phase extraction-based
platform for rapid phosphoproteomic analysis. Methods 2011, 54,
379386.
(29) Lin, Y.; et al. Sodium-deoxycholate-assisted tryptic digestion and
identification of proteolytically resistant proteins. Anal. Biochem. 2008,
377, 259266.
(30) Leon, I. R.; et al. Quantitative assessment of in-solution
digestion efficiency identifies optimal protocols for unbiased protein
analysis. Mol. Cell. Proteomics 2013, 12, 29923005.

scale study of protein regulation controlled by the interplay of

several phosphorylation sites.

ASSOCIATED CONTENT

* Supporting Information
S

In addition to supporting Figure S1, three supporting tables

with identied phosphopeptides (Supporting Table S1
[replicate experiment], Supporting Table S2 [ERLICSCX
SPE], and Supporting Table S3 [down scaling of SCXSPE])
are submitted with this manuscript. This material is available
free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Downloaded by NATL LBRY OF SERBIA on September 11, 2015 | http://pubs.acs.org

Publication Date (Web): November 4, 2013 | doi: 10.1021/pr4007969

Corresponding Authors

*J.D.: Tel: +49-761-203-97208. Fax: +49-761-203-8456. Email: joern.dengjel@frias.uni-freiburg.de.

*M.Z.: Tel: +49-761-203-97220. E-mail: mostafa.zarei@frias.
uni-freiburg.de.
Author Contributions

M.Z. and A.S. contributed equally to this work.

Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
The research leading to these results has received funding from
the Excellence Initiative of the German Federal and State
Governments through FRIAS and BIOSS, from the Federal
Ministry of Education and Research through GerontoSys II
_NephAge (031 5896 A), and from the German Research
Foundation, DFG (DE 1757/2-1). We thank Eksigent/AB
Sciex for their generous gift of a 2D nanoLC and Remco van
Soest for his technical advice for maintenance and trouble
shooting.

REFERENCES

(1) Hunter, T. The age of crosstalk: phosphorylation, ubiquitination,

and beyond. Mol. Cell 2007, 28, 730738.
(2) Johnson, L. N.; Barford, D. The effects of phosphorylation on the
structure and function of proteins. Annu. Rev. Biophys. Biomol. Struct.
1993, 22, 199232.
(3) Halbach, S.; et al. Alterations of Gab2 signalling complexes in
imatinib and dasatinib treated chronic myeloid leukaemia cells. Cell
Commun. Signaling 2013, 11, 30.
(4) Olsen, J. V.; et al. Global, in vivo, and site-specific
phosphorylation dynamics in signaling networks. Cell 2006, 127,
635648.
(5) Boersema, P. J.; Mohammed, S.; Heck, A. J. R. Phosphopeptide
fragmentation and analysis by mass spectrometry. J Mass Spectrom
2009, 44, 861878.
(6) Schroeder, M. J.; Shabanowitz, J.; Schwartz, J. C.; Hunt, D. F.;
Coon, J. J. A neutral loss activation method for improved
phosphopeptide sequence analysis by quadrupole ion trap mass
spectrometry. Anal. Chem. 2004, 76, 35903598.
(7) Jedrychowski, M. P.; et al. Evaluation of HCD- and CID-type
fragmentation within their respective detection platforms for murine
phosphoproteomics. Mol. Cell Proteomics 2011, 10, M111.009910.
(8) Marx, H.; et al. A large synthetic peptide and phosphopeptide
reference library for mass spectrometry-based proteomics. Nat.
Biotechnol. 2013, 31, 557564.
(9) Andersson, L.; Porath, J. Isolation of phosphoproteins by
immobilized metal (Fe3+) affinity chromatography. Anal. Biochem.
1986, 154, 250254.
5994

dx.doi.org/10.1021/pr4007969 | J. Proteome Res. 2013, 12, 59895995

Journal of Proteome Research

Technical Note

Downloaded by NATL LBRY OF SERBIA on September 11, 2015 | http://pubs.acs.org

Publication Date (Web): November 4, 2013 | doi: 10.1021/pr4007969

(31) Sugiyama, N.; et al. Phosphopeptide enrichment by aliphatic

hydroxy acid-modified metal oxide chromatography for nano-LC-MS/
MS in proteomics applications. Mol. Cell Proteomics 2007, 6, 1103
1109.
(32) Cox, J.; Mann, M. MaxQuant enables high peptide identification
rates, individualized p.p.b.-range mass accuracies and proteome-wide
protein quantification. Nat. Biotechnol. 2008, 26, 13671372.
(33) Cox, J.; et al. Andromeda: a peptide search engine integrated
into the MaxQuant environment. J. Proteome Res. 2011, 10, 1794
1805.
(34) Frese, C. K.; et al. Unambiguous phosphosite localization using
electron-transfer/higher-energy collision dissociation (EThcD). J.
Proteome Res. 2013, 12, 15201525.
(35) Kim, M.-S.; Zhong, J.; Kandasamy, K.; Delanghe, B.; Pandey, A.
Systematic evaluation of alternating CID and ETD fragmentation for
phosphorylated peptides. Proteomics 2011, 11, 25682572.

5995

dx.doi.org/10.1021/pr4007969 | J. Proteome Res. 2013, 12, 59895995