Protein Phosphorylation and Expression Profiling by Yin-Yang
Multidimensional Liquid Chromatography (Yin-Yang MDLC) Mass
Spectrometry
Jie Dai, Wen-Hai Jin, Quan-Hu Sheng, Chia-Hui Shieh, Jia-Rui Wu, and Rong Zeng*
Research Center for Proteome Analysis, Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Received August 16, 2006
A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with
mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange
(SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX
column. The flow-through peptides from SCX were collected and further loaded on an SAX column.
Both columns were eluted by offline pH steps, and the collected fractions were identified by reversephase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was
achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were
identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with
809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features
of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass
spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated
peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134
groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but
different phosphorylated states. This unbiased profiling of protein expression and phosphorylation
provides a powerful approach to probe protein dynamics, without using any prefractionation and
chemical derivation.
Keywords: Protein phosphorylation • Protein expression • Strong-cation exchange • Strong-anion exchange • YinYang multidimensional liquid chromatography • pH elution • Mass spectrometry
Introduction
The proteins in cells behave in a very dynamic manner,
which includes the alteration of protein expression, protein
modification, protein distribution, and interaction. When a cell
receives a stimulus from the environment, one of the ubiquitous cellular responses includes protein phosphorylation/
dephosphorylation. The phosphorylation behavior frequently
triggers signal transduction, followed by gene regulation and
protein expression.1-4 Therefore, the protein phosphorylation
and expression appear to be the most important targets
analyzed by current proteomics. Many large-scale protein
identification methods were recently developed to probe the
protein expression profile.5-8 On the other hand, the efforts to
elucidate the phosphorylation seem more difficult due to the
low sensitivity to phosphorylated peptides in mass spectrometry.4
Current global analysis of protein phosphorylation requires
an enrichment method to increase detection of low-abundance
* To whom correspondence should be addressed at Research Center for
Proteome Analysis, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200031,China.Tel.: 86-21-54920170.Fax: 86-21-54920171;E-mail: [email protected].
250
Journal of Proteome Research 2007, 6, 250-262
Published on Web 12/14/2006
phosphoproteins. Some high-affinity chemical derivation
methods15-18 have emerged, but suffer from side reactions,
marginal yields, and low specificities. Immunopurification19-22
and affinity chromatography including immobilized metal
affinity chromatography (IMAC) and titanium dioxide (TiO2)
are frequently used techniques for the enrichment of phosphorylated proteins and peptides,23-28 but also suffer from
unsatisfactory specificity or sensitivity. Recently, strong-cation
exchange (SCX) chromatography using salt gradient has been
applied for phosphopeptide enrichment by collecting fractions
from an SCX column followed by reverse-phase chromatography and mass spectrometry.29,30
Combination of different separation or HPLC methods has
also been used to decrease the complexity of proteins and
increase the concentration of phosphopeptides. First, proteins
were subjected to a prefractionation, such as gel electrophoresis, before the protein digestion.29,30 Some groups combined
two affinity methods for phosphopeptides such as antibody
purification combined with IMAC,19 or ion-exchange chromatography and IMAC,31,32 to increase the capacity of identified
phosphopeptides. Subcellular fractionation was also frequently
contributed to decrease the protein complexity.29,31,32
10.1021/pr0604155 CCC: $37.00
 2007 American Chemical Society
Protein Phosphorylation/Expression Profiling by Yin-Yang MDLC
In our recent work, we used the pH gradient in the SCX to
replace the traditional salt elution, which demonstrated the
distribution of peptides roughly according to their pI values.33
In this report, we designed an effective way to obtain the total
information of peptides, using the newly developed Yin-Yang
multidimensional liquid chromatography tandem mass spectrometry (Yin-yang-MDLC-MS/MS) method. This method
combines SCX, strong-anion exchange (SAX), and reverse-phase
separation for the comprehensive protein identification. The
SCX and SAX in this system displayed complementary acquisition of peptides. The SAX-based method can obtain more
extremely acidic peptides (pI < 4.0) and phosphopeptides,
while SCX-based method identifies relatively basic peptides (pI
> 4.0). In total, 134 groups of phosphorylated peptide isoforms
were observed in this data set, which have common peptide
sequences but different phosphorylated states. This method
provided an unbiased profiling of protein expression and
phosphorylation, without introducing any prefractionation and
chemical derivation.
Experimental Procedures
Chemicals. All the water used in this experiment was
prepared using a Milli-Q system (Millipore, Bedford, MA).
Dithiothreitol (DTT), ammonium bicarbonate, and iodoacetamide (IAA) were all purchased from Bio-Rad (Hercules, CA).
Urea was obtained from Sigma (St. Louis, MO). Trypsin was
purchased from Promega (Madison, WI). Formic acid (FA) was
obtained from Aldrich (Milwaukee, WI). Acetonitrile (HPLC
gradient grade) was obtained from Merck (Darmstadt, Germany). Sodium orthovanadate (Na3VO4) and sodium fluoride
(NaF) were obtained from Sigma (St. Louis, MO); ethylene
diamine tetraacetic acid (EDTA), ethylene glycol-bis-[2-aminoethyl ether]-N,N,N′,N′ tetraacetic acid (EGTA), and phenyl
methyl sulfonyl fluoride (PMSF) were purchased from Amresco
(Solon, OH). All the chemicals are of analytical grade except
acetonitrile, which is of HPLC grade. The SCX column (0.32 ×
100 mm), SAX column (0.32 × 100 mm), and pH buffers kit
were bought from Column Technology, Inc., (Fremont, CA).
Mouse Liver Sample Preparation. Mice were sacrificed, and
their livers were promptly removed and placed in an ice-cold
homogenization buffer consisting of 8 M urea, 4% CHAPS (w/
v), 65 mM DTT, 1 mM EDTA, 0.5 mM EGTA, and a mixture of
protease inhibitor (1 mM PMSF), phosphatase inhibitors (0.2
mM Na3VO4 and 1 mM NaF), and 40 mM Tris-HCl at pH 7.4.
After they were minced with scissors and washed to remove
blood, the livers were homogenized in a Potter-Elvejhem
homogenizer with a Teflon piston, using 10 mL of the homogenization buffer per 2 g of tissue. The suspension was homogenized for approximately 1 min, sonicated for 100 W × 30 s
and centrifuged at 25 000g for 1 h. The supernatants were
colleted.
Tryptic Digestion of Protein Mixture. The tryptic digestion
was processed according to a previously published method.33
Briefly, 1 mg of proteins was redissolved in reducing solution,
then mixed with 5 µL of 1 M DTT. The mixture was incubated
at 56 °C for 1 h. Forty microliters of 1 M IAA was then added,
and the mixture was incubated for an additional 40 min at
room temperature in darkness. The protein mixtures were spun
and exchanged into 100 mM ammonium bicarbonate buffer,
and then incubated with trypsin (25:1) at 37 °C for 20 h.
Yin-Yang-MDLC-MS/MS System. The SCX and SAX columns were rinsed before sample loading. Finally, the SCX
column was filled with a pH 2.5 buffer, and the SAX column
research articles
was filled with a pH 10.0 buffer. In this study, a series of elution
buffers with different pH (pH 10.0, 8.5, 8.0, 7.0, 6.0, 5.5, 5.0,
4.5, 4.0, 3.5, 3.0, and 2.5,) were used for pH step gradient
elution. First, a total of 1 mg of peptide mixture was lyophilized,
and then dissolved in 100 µL of pH 2.5 buffer. Then, the sample
was injected into the SCX column by a syringe pump, at a flow
rate of 3 µL/min. Then, the column was flushed with another
100 µL of elution buffer at pH 2.5. The flow-through fraction
was collected and lyophilized for SAX column fractionation.
Thereafter, the SCX column was eluted by 200 µL each of
buffers at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 8.5, and
10.0 sequentially, and the corresponding eluted fraction was
collected individually and lyophilized for nanoLC-MS. The
unbound fraction at pH 2.5 of the SCX column was lyophilized
and dissolved in 100 µL of pH 10.0 buffer, then loaded into the
SAX column. Then, another 100 µL of pH 10.0 buffer was used
to elute the unbound peptide mixture in the SAX column. The
flow-through peptide mixture was collected. The SAX column
was eluted by 200 µL each of buffers at pH 8.5, 8.0, 7.0, 6.0,
5.5, 5.0, 4.5, 4.0, 3.5, 3.0, and 2.5 sequentially, and the corresponding eluted fraction was collected individually and lyophilized for nanoLC-MS.
A Surveyor liquid chromatography system (Thermo Finnigan,
San Jose, CA), consisting of a degasser, MS Pump, and autosampler, and equipped with a C18 trap column (RP, 320 µm
× 20 mm, Column Technology, Inc., Fremont, CA) and an
analytical C18 column (RP, 75 µm × 150 mm, Column
Technology, Inc., Fremont, CA) was used. The HPLC solvents
used were 0.1% formic acid (v/v) aqueous (A) and 0.1% formic
acid (v/v) acetonitrile (B). The samples were loaded into the
trap column first at a 3 µL/min flow rate after the split, and
then the reverse-phase gradient was from 2 to 40% mobile
phase B in 165 min at 100 µL/min flow rate before the split
and 250 nL/min after the split. A Finnigan LTQ linear ion trap
mass spectrometer equipped with a nanospray source was used
to for the MS/MS experiment with ion transfer capillary at 160
°C and NSI voltage of 1.8 kV. Normalized collision energy was
35.0. The mass spectrometer was set so that one full MS scan
was followed by 10 MS/MS scans with the following Dynamic
Exclusion settings: repeat count 2, repeat duration at 30 s,
exclusion duration at 90 s.
Data Analysis and Validation. All .dta files were created
using Bioworks 3.2, with precursor mass tolerance of 1.4 Da,
threshold of 100, and minimum ion count of 15. The acquired
MS/MS spectra were searched against the Mouse International
Protein Index protein sequence database (version 3.07, www.ebi.ac.uk/IPI) combined with real protein and reverse sequences of proteins, by using the TurboSEQUEST program in
the BioWorks 3.2 software suite, with a mass tolerance of 3.0
Da. One missed cleavage site of trypsin was allowed. Dynamic
modifications were allowed for the detection of oxidized Met
(+16), and phosphorylated Ser, Thr, and Tyr (+80).
All output results were combined using an in-house software
named BuildSummary. When no modifications were included,
the filter was set to Xcorr g1.9 with charge state of 1+, Xcorr
g2.2 with charge state of 2+, and Xcorr g3.75 with charge state
of 3+; ∆Cn g 0.1. For phosphopeptides, the filter was set to
Xcorr g2.0 with charge state of 1+, Xcorr g2.5 with charge state
of 2+, and Xcorr g3.3 for charge state of 3+. Both of the filters
used ∆Cn above 0.1. The unmodified peptide and phosphopeptide were combined to assign proteins. Finally, only proteins
with at least two unique peptides were kept. For proteins that
have single phosphopeptides identified, only phosphopeptides
Journal of Proteome Research • Vol. 6, No. 1, 2007 251
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Dai et al.
Figure 1. The flowchart of the Yin-yang-MDLC-MS/MS. Protein digests were loaded on the SCX column first, and the flow-through
fraction was collected. The bound peptides in SCX were eluted by 11-pH steps from pH 3.0 to pH 10.0. The flow-through fraction was
loaded on the offline SAX column and eluted from pH 10.0 to pH 2.5. Each fraction from SCX and SAX was identified by reverse-phase
liquid chromatography coupled with mass spectrometry.
with at least two MS/MS spectra were kept. The false-positive
rate was calculated based on the following formula: % fal )
2[nrev/(nrev + nreal)]. The nrev is the number of peptide hits
matched to “reverse” protein, and nreal is the number of peptide
hits matched to “real” protein.8 The false-positive rate was
finally 1.3%. Further manual check removed the suspicious
phosphopeptides with unclear MS/MS spectra. Therefore, the
final false-positive rate of this data set should be <1.3%.
For phosphorylated site determination, when an exact phosphorylated site could not be determined in a phosphorylated
peptide, the site was defined as ambiguous. If ambiguous sites
in one MS spectrum were confirmed by exact sites in another
MS spectrum, the ambiguous sites would not be counted. If
peptides have a common sequence, but different numbers of
ambiguous phosphorylated sites, the least ambiguous sites were
counted.
Results and Discussion
Peptide and Phosphopeptide Identification by Yin-Yang
MDLC Method. A Yin-yang-MDLC-MS/MS system was constructed based on the pH elution. Figure 1 shows the flowchart
of the Yin-yang-LC system. One milligram of mouse liver
protein digest was loaded onto the SCX column first, and the
flow-through fraction was collected. The bound peptides in the
SCX were eluted by 11-pH steps from pH 3.0 to pH 10.0. The
flow-through fraction was loaded on the offline SAX column
and eluted by 12 steps from pH 10.0 to pH 2.5. Each fraction
from the SCX and SAX was loaded on reverse-phase coupled
with mass spectrometry. In this method, the basic peptides
were retained and separated by strong-cation exchange column. The acidic peptides which did not bind to the SCX
column was collected and separated by anion exchange
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Journal of Proteome Research • Vol. 6, No. 1, 2007
column. This is particularly important for the recovery of
phosphopeptides which are more acidic than other peptides
in general. All the fractions from both columns then undergo
reverse-phase separation followed by tandem mass spectrometry. This is the most comprehensive identification of the
peptides and phosphopeptides in the MDLC-MS/MS. All the
peptides were either retained or separated by the strong-cation
exchange, or by the anion exchange and then followed by
reverse-phase chromatography. This design can be used to
identify both pospho- and nonphosphopeptides in one analysis.
Table 1 shows the mouse liver proteome identified in Yinyang MDLC mass spectrometry, including the unmodified and
phosphorylated peptide identification in each SCX and SAX
fraction, respectively. Totally, 9971 unique unmodified peptides
were identified in SCX-RP-MS, while 6403 were identified in
SAX-RP-MS, resulting in 13 256 unique unmodified peptides.
On the other hand, 659 unique phosphopeptides were detected
in SAX-RP-MS, and 210 phosphopeptides were identified in
SCX-RP-MS. Twenty phosphopeptides were both identified
by SAX-RP-MS and SCX-RP-MS. The combined 849 unique
phosphopeptides were identified including 809 phosphorylated
sites by the Yin-yang-MDLC-MS/MS system, under the low
false-positive rate of 1.3%. Of the 809 phosphorylated sites,
there are 607 unique sites, of which 425 (70.02%) are on Ser,
130 (21.42%) on Thr, and 52 (8.57%) on Tyr. The other 202
phosphorylated sites belong to 458 ambiguous candidates. In
SAX-RP-MS, 2.95 spectra were obtained on average for each
phosphopeptides, while in SCX-RP-MS, the ratio was only
1.59. The ratios for unmodified peptides are very similar in SAX
(9.80) and SCX (8.18). The higher number of MS/MS spectra
indicates the higher intensity of the phosphopeptides in SAX.
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Protein Phosphorylation/Expression Profiling by Yin-Yang MDLC
Table 1. The Unmodified Peptides and Phosphorylated Peptides Identified in Each Fraction by SCX-RP-MS (A and B) and
SAX-RP-MS (C and D)
fraction
pH 3.0
pH 3.5
unique peptides
peptide hits
ratio (hits/unique)
3677
15448
4.201
3778
17552
4.646
unique peptides
peptide hits
ratio (hits/unique)
70
99
1.414
62
94
1.516
fraction
pH 2.5
pH 3.0
unique peptides
peptide hits
ratio (hits/unique)
698
2010
2.880
471
2092
4.442
unique peptides
peptide hits
ratio (hits/unique)
78
249
3.192
110
393
3.573
pH 4.0
pH 4.5
pH 5.0
pH 5.5
pH 6.0
(A) Unmodified Peptides Identified in SCX
2342
1829
1539
1270
801
10552
9876
8472
5889
3226
4.506
5.400
5.505
4.637
4.027
(B) Phosphopeptides Identified in SCX
35
22
10
16
5
38
26
19
26
6
1.086
1.182
1.900
1.625
1.200
pH 3.5
pH 4.0
pH 4.5
pH 5.0
pH 5.5
(C) Unmodified Peptides Identified in SAX
588
648
862
699
955
3256
5132
3921
3490
4226
5.537
7.920
4.549
4.993
4.425
(D) Phosphopeptides Identified in SAX
186
91
54
28
59
407
174
94
75
110
2.188
1.912
1.741
2.679
1.864
The Yin-yang MDLC system used the SCX as the first
dimension, and the unbound materials were further loaded and
separated using SAX. The coupling of SCX and SAX captured
peptides more comprehensively than SCX alone. As our results
show, the flow-though fraction still contained thousands of
peptides, especially many phosphopeptides. In reported work,
fractions from SCX column with salt gradient in pH 2.729,30 were
collected and followed by mass spectrometry identification, and
6 mg of protein fractionated by SDS-PAGE were needed before
the SCX separation of peptides. Other work combined the ion
exchange with IMAC to increase the sample capacity.31,32 Our
method is much simpler and easier to handle based only on
ion exchange chromatography, consuming only 1 mg of
proteins. Unlike other enrichment methods of phosphopeptides, this method obtained many phosphopeptides without
loss of unmodified peptides. The identified unmodified peptides and phosphopeptides were listed in Supplementary Tables
1 and 2 of Supporting Information.
SCX and SAX Coupled with Mass Spectrometry Can Identify
Different Peptides. Figure 2 shows the base peak of fractions
in SAX-RP-MS. The clear peaks in the acidic regions indicated
that many acidic peptides were identified by the SAX-RP-MS.
Figure 3 presents the distribution of the theoretical pI of the
identified unmodified peptides. It is very interesting that the
SCX-RP-MS and SAX-RP-MS identification are extremely
complementary. In SCX-RP-MS, many more peptides with pI
g 4.0 were identified than in SAX-RP-MS. However, for the
unmodified peptides with theoretical pI 3.5-4.0, 6944 spectra
were obtained for 796 unique peptides identified by SCX-RPMS, while up to 35 884 spectra were identified for 2174 unique
peptides in SAX-RP-MS. For peptides with pI 3.0-3.5, only 25
spectra for 9 unique peptides were obtained in SCX-RP-MS,
but SAX-RP-MS still identified 1360 spectra belonging to 96
unique peptides. Thus, the SAX-RP-MS identified 5.34-fold
more of the spectra and 2.82-fold more of the unique peptides
than SCX-RP-MS, for the unmodified peptides with theoretical pI < 4.0.
Figure 4 illustrates the theoretical pI distribution of the
nonphosphorylated precursors of the identified singly phosphorylated peptides in SCX and SAX. Most of the peptide
backbones of the phosphopeptides were originally acidic with
the pI below 4.5. For peptides with pI 4.0-4.5, 144 singly
phosphorylated products were detected, in which 40 unique
pH 7.0
pH 8.0
pH 8.5
pH 10.0
1098
3970
3.616
553
1643
2.971
556
1674
3.011
562
3284
5.843
6
10
1.667
0
0
/
1
1
1.000
11
12
1.091
pH 6.0
pH 7.0
pH 8.0
pH 8.5
pH 10.0
972
5428
5.584
1544
7257
4.700
1615
8811
5.456
3485
10804
3.100
2330
6351
2.726
50
104
2.080
68
118
1.735
55
95
1.727
50
65
1.300
22
27
1.227
peptides (55 hits) were detected in SCX-RP-MS, and 104
unique peptides (181 hits) were found in SAX-RP-MS. For
peptides with pI 3.5-4.0, 344 unique peptides (with 1222 hits)
were detected with their singly phosphorylated peptides by
SAX-RP-MS, while only 10 of them (with 19 hits) were
identified in SCX-RP-MS. It is interesting that SCX-RP-MS
lost most of the unmodified peptides with pI below 4.0 (as
Figure 3). SCX-RP-MS also cannot identify most of the
phosphorylated peptides if their precursor pI values were below
4.5. It seems that the single-phosphorylation statistically decreased the pI of the peptides 0.5 unit. Figure 4A also indicates
that most of the nonphosphorylated precursors of the phosphopeptides are originally acidic with pI below 4.5. Thus, the
phosphorylation will make the peptides more acidic, which
further increases their loss in the SCX-RP-MS, and these were
then identified in the secondary SAX-RP-MS. Most of the
multiple phosphorylated peptides (82% of the peptide hits)
were detected in SAX-RP-MS. The movement of the pI to more
acidic regions by multiphosphorylation leads to further loss in
SCX-RP-MS. It is also notable that there are still some
phosphopeptides obtained by SCX-RP-MS, including 69
phosphorylated peptides that their nonphosphorylated precursors were originally basic, such as >8.0. Therefore, the phosphopeptide distribution is more comprehensive than expected.
The SCX-RP-MS preferentially identified some phosphopeptides if their nonphosphorylated precursors are very basic. The
phosphorylation did not make them too acidic to be retained
by SCX-RP-MS. The 849 unique phosphorylated peptides
were listed in Supplementary Table 2 of Supporting Information.
Different Distribution Tendency of Peptides in SCX and
SAX. Figure 5 illustrates the phosphopeptide and unmodified
peptide distribution profiles in the SCX-RP-MS and SAX-RPMS. In SCX-RP-MS, the phosphopeptides and unmodified
peptides display the similar distribution tendency along with
the pH increase. The number of these two kinds of peptides
both declined when pH increased. In contrast, in SAX-RP-MS,
the phosphopeptides and unmodified peptides had opposite
focusing tendency. When pH decreased, the level of unmodified
peptides declined significantly, while the phosphopeptides
increased. In the SCX, even in the acidic region, the phosphorylated peptides still mixed with many unmodified peptides.
This indicates, though SCX might retain some phosphopepJournal of Proteome Research • Vol. 6, No. 1, 2007 253
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Dai et al.
Figure 2. The base peak of SAX-RP-MS in the Yin-yang MDLC-MS/MS system. More intensive signals were obtained in acidic
regions.
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Journal of Proteome Research • Vol. 6, No. 1, 2007
Protein Phosphorylation/Expression Profiling by Yin-Yang MDLC
research articles
Figure 3. The distribution of unique peptides (A) and spectra hits (B) from unmodified peptides, in given theoretical pI regions. The
gray bars show the identified numbers in SCX-RP-MS, and black bars represent the identified numbers in SAX-RP-MS. The X-axis
represents theoretical pI range calculated from unmodified peptides, and the Y-axis shows the numbers of peptides and spectra hits
in each pI range.
tides, the coelution of unmodified peptides with phosphopeptides in acidic or low-salt regions still suppressed the identification of phosphopeptides by mass spectrometry. Therefore,
the secondary IMAC was used to further enrich the phosphopeptides in previous work.31 Otherwise, prefractionation of
proteins before SCX was used to decrease the coeluted,
unmodified peptides in low-salt region.29,30 In this work, the
SAX-RP-MS seems to be more suitable to enrich and identify
phosphopeptides than SCX-RP-MS. The only concern of the
SAX is the limited loading capacity. Therefore, the use of SCX
prior to SAX bound most unmodified peptides and significantly
decreased the complexity of the peptide mixture. The phosphopeptides were further focused to acidic regions in SAX by
pH elution, which decreased the suppression of nonphosphopeptides to phosphopeptides as much as possible. This SCXSAX mode can identify more phosphopeptides than SAX-only
mode, or SAX-SCX mode (data not shown).
In the offline mode, the traditional salt elution needs
desalting, resulting in sample loss. Because of the strong
binding of the phosphopeptides with the SAX, the phosphopeptides need to be eluted by high concentration of salt if the
traditional mode is used. This may be problematic for the
analysis of phosphopeptides, because phosphopeptides are
more likely to be lost during the desalting process, as reported
by several groups.32,34-37 For a pH gradient-based ion exchange
chromatography, the basic peptides will elute at higher pHs
and the acidic peptides will elute at lower pHs, with the static
low concentration (5-10 mM) of salt as we reported.33 The pH
elution had excellent peptide resolution according to the
peptide pI.33 The pH step elution method used in this study
not only enriched the phosphopeptides to acidic regions, but
also reduced the risk of phosphopeptide losses to a minimum,
since no further desalting was needed.
Comprehensive Coverage of Peptides and Proteins of the
Yin-Yang MDLC-MS/MS. This unbiased method based on the
Yin-Yang system can achieve more complete resolution of
phosphopeptides, as well as unmodified peptides, than traditional methods. Among the 13 256 unique and unmodified
peptides, only 3118 were identified both by SCX-RPLC-MS
and SAX-RPLC-MS, demonstrating 23.5% overlapping. Of the
849 unique phosphopeptides, only 20 were identified both by
SCX and SAX. When the unmodified and phosphopeptides are
combined, 14 105 unique peptides were identified, corresponding to 2804 high-confidence identifications of proteins. There
are 72.2% of proteins that were identified both by SCX and SAX
(2024 overlapped protein vs 2804 total proteins), which indicated more peptide coverage of the proteins was obtained from
multiple peptide matching, due to the complementary features
of SCX-RP-MS and SAX-RP-MS. SCX-RP-MS occupied
75.2% unique unmodified peptides and SAX-RP-MS identified
Journal of Proteome Research • Vol. 6, No. 1, 2007 255
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Dai et al.
Figure 4. The distribution of unique peptides (A) and spectra hits (B) from nonphosphorylated precursors of the phosphopeptides, in
given theoretical pI regions. The gray bars show the identified numbers in SCX-RP-MS, and black bars represent the identified numbers
in SAX-RP-MS. The X-axis shows the theoretical pI range calculated from nonphosphorylated precursors of the phosphopeptides,
and the Y-axis demonstrates the numbers of peptides and spectra hits in each pI range.
77.6% of the unique phosphopeptides. The Yin-Yang-MDLCMS/MS system shows complementarity and high resolution for
proteome analysis. For example, protein alpha-1 catenin was
identified with 13 unmodified peptides and 2 phosphopeptides,
in which 11 unmodified peptides were only found in SCXRP-MS, 1 found only in SAX-RP-MS, and 1 detected both in
SAX and SCX. The two phosphopeptides were only detected
by SAX-RP-MS. Table 2 shows the distribution of peptides
from alpha-1 catenin. The Yin-Yang-MDLC-MS/MS system
provides an unbiased method to obtain phosphopeptides, as
well as unmodified peptides. Certainly, when only phosphopeptides were focused, this method can be combined with
other affinity methods to further enrich the phosphopeptides.
In this work, extensive manual check was needed, especially
for the phosphopeptides, due to the low-resolution of the linear
ion trap mass spectrometry. Further work using high-resolution
LTQ-Orbitrap mass spectrometry has shown to be more
powerful for phosphopeptide identification (Dai, J. et al.,
manuscript in preparation).
Peptide Isoforms Obtained in Yin-Yang-MDLC-MS/MS.
It is interesting that phosphopeptide isoforms with common
peptide sequence but different phosphorylated states can be
obtained by this method. There are 134 groups of phosphopeptide isoforms acquired by this method. It is noted that most
of the isoform groups are identified by SAX-RP-MS. Among
the 134 phosphopeptide isoforms, 101 groups with all compo256
Journal of Proteome Research • Vol. 6, No. 1, 2007
nents were only found in SAX-RP-MS, 3 groups only found
in SCX-RP-MS, and 30 groups were found complementary
in SCX-RP-MS and SAX-RP-MS.
Panels A and B of Figure 6 show the spectra of peptide
isoforms of DQWGSEEEEEAGGYR and DQWGS*EEEEEAGGYR,
respectively. These two groups of peptides both belong to the
Cdc42 effector protein 4, which was not found with phosphorylation before in any reference or annotation (www.expasy.ch).
The nonphosphopeptide shows the dominant y13 ion, while
the phosphopeptide has the intensive peak form neutral loss.
However, the common y3 to y10 confirmed the phosphopeptide identification.
Figure 6C,D shows the mass spectra of peptide isoforms of
VPS(*)S(*)DEEVVEEPQSR, with single but ambiguous phosphorylation, and VPS*S*DEEVVEEPQSR, with double distinct
phosphorylated sites, both found in SAX-RP-MS. In the
monophosphopeptide spectra, the y4 and y7 are still dominant.
In the doubly phosphopeptide spectra, the y4 and y7 are
suppressed by the neutral loss peak. The serial y3-y11 confidently identifies the doubly phosphorylated peptide. These
phosphopeptides belong to the mKIAA1741 protein, which is
annotated as 182 kDa tankyrase 1-binding protein (http://
harvester.embl.de). The same phosphorylated sites were also
found most recently.31
Figure 6E,F presents the spectra of peptide isoforms of
GDKS(*)S(*)EPTEDVETK, with a monophosphorylated site
research articles
Protein Phosphorylation/Expression Profiling by Yin-Yang MDLC
Figure 5. The distributions of phosphopeptides and unmodified peptides in fractions of SCX-RP-MS (A) and SAX-RP-MS (B). The
X-axis shows the pH fractions in each method, and the Y-axis presents the percentage of identified spectra hits in each fraction vs all
spectra hits in each method.
Table 2. The Distribution of Unmodified Peptides and Phosphpeptides in SCX-RP-MS and SAX-RP-MS, from Protein Alpha-1
Catenina
IPI:IPI00112963.1| Alpha-1 catenin
fraction
in SCX
(pH)
charge
pI
K.AHVLAASVEQATENFLEK.G
K.AVMDHVSDSFLETNVPLLVLIEAAK.N
K.HVNPVQALSEFK.A
K.IAEQVASFQEEK.S
K.NVPILYTASQACLQHPDVAAYK.A
K.QIIVDPLSFSEER.F
K.SAAGEFADDPCSSVK.R
K.SKLDAEVSK.W
K.SQGMASLNLPAVSWK.M
R.MSASQLEALCPQVINAALALAAKPQSK.L
R.QALQDLLSEYMGNAGR.K
R.TpSpVQTpEDDQLIAGQSAR.A
2
2 and 3
2
2
2
2
2
2
2
3
2
2
4.75
4.31
6.75
4.25
6.73
4.14
4.03
5.79
8.47
7.95
4.37
4.03
5
4.5
5.5
3
4
3.5
3
5
3
4.5
3.5
N.F
R.TPEELDDS*DFETEDFDVR.S
R.TPEELDDSDFETEDFDVR.S
R.VLTDAVDDITSIDDFLAVSENHILEDVNK.C
2
2
3
3.51
3.51
3.76
N.F
N.F
3.5, 4.0, 4.5,
6.0, 7.0
fraction
in SAX
(pH)
N.F
N.F
N.F
8.5
N.F
N.F
N.F
N.F
N.F
N.F
N.F
4.5, 5.0,
5.5, 6.0
2.5
3
N.F
modified
site
N.F
N.F
N.F
N.F
N.F
N.F
N.F
N.F
N.F
N.F
N.F
2
1
N.F
N.F
N.F
a
(*) Indicates the phosphorylated site is unique; p indicates the phosphorylated site is ambiguous; N.F., not found.
ambiguously on serine, and GDKS*S*EPTEDVETK, with phosphorylation on both serine residues. The doubly phosphorylated spectrum has a more complicated spectra and is not easily
identified. The series of y3-y5 ions and the obvious y8 ions
that appeared in both peptide isoforms display similar patterns
of fragmentations, confirming the identification of the multiphosphorylated peptide. These peptides were from the TransGolgi network integral membrane protein 1. This protein is a
membrane protein located in the golgi apparatus and has been
thought of as a glycoprotein. The phosphorylation of this
protein was first found in this work.
Supplementary Table 3 in Supporting Information shows the
134 peptide groups with nonphosphopeptides, monophosphopeptides, and multiphosphopeptides. Almost all of the phosphorylated peptides are eluted in more acidic fractions than
their nonphosphorylated counterparts. Also, more phosphopeptide isoforms were identified by SAX-RP-MS. It is reasonable to expect that, if a peptide has multiple potential phosJournal of Proteome Research • Vol. 6, No. 1, 2007 257
research articles
Figure 6 (continued)
258 Journal of Proteome Research • Vol. 6, No. 1, 2007
Dai et al.
Protein Phosphorylation/Expression Profiling by Yin-Yang MDLC
research articles
Figure 6 (continued)
Journal of Proteome Research • Vol. 6, No. 1, 2007 259
research articles
Dai et al.
Figure 6. The MS/MS spectra of representive peptide isoforms: peptide DQWGSEEEEEAGGYR (A) and its monophosphorylated peptide
DQWGS*EEEEEAGGYR (B); monophosphopeptide VPS*SDEEVVEEPQSR (C) and its doubly phosphorylated peptide VPS*S*DEEVVEEPQSR(D); monophosphopeptide GDKS(*)S(*)EPTEDVETK (E) and corresponding doubly phosphorylated peptide GDKS*S*EPTEDVETK (F).
The (*) means ambiguous phosphorylated site, and the * means distinct phosphorylated site.
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Protein Phosphorylation/Expression Profiling by Yin-Yang MDLC
phorylated sites, more serines and threonines will be contained.
The phosphorylation will make them more acidic, with makes
it much easier to be lost in SCX-RP-MS. Supplementary Table
4 in Supporting Information shows the identification details
of all unmodified peptides and phosphopeptides.
Conclusion
In conclusion, this methodology significantly enhances the
ability to routinely discover protein expression and phosphorylation within very complex protein mixtures by exploiting pH
step-based Yin-yang-MDLC-MS/MS. The pH elution enables
the sensitive identification and minimum sample loss of
phosphopeptides. The separation and identification of phosphopeptides using SAX-RP-MS also presents a novel strategy
for phosphoproteomics. The combination of the SCX-RP-MS
and SAX-RP-MS achieved comprehensive identification of
phosphorylated as well as unmodified peptides.
Phosphoproteins can exist with varying degrees of phosphorylation, reflecting different functional states of cells. Also, the
phosphorylation will trigger gene expression leading to alteration of protein expression. Further work is ongoing that
combines this Yin-yang-MDLC-MS/MS with quantitative
methods, such as iTRAQ and SILAC,10,11,39 to probe the dynamics of the protein expression as well as phosphorylation.
Abbreviations: SAX, strong-anion exchange; SCX, strongcation exchange; Yin-yang MDLC-MS/MS, Yin-Yang multidimensional liquid chromatography tandem mass spectrometry;
IMAC, immobilized metal affinity chromatography; TiO2, titanium dioxide; LC, liquid chromatography; SCX-RP-MS, strongcation exchange reverse-phase liquid chromatography mass
spectrometry; SAX-RP-MS, strong-anion exchange reversephase liquid chromatography mass spectrometry; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; EDTA,
ethylene diamine tetraacetic acid; EGTA, ethylene glycol-bis[2-aminoethyl ether]-N,N,N′,N′ tetraacetic acid.
Acknowledgment. We thank Mr. Lei Zhang of Shanghai
Institute of Biochemistry and Cell Biology for his maintenance
of the PC cluster system. This work was supported by the
National Natural Science Foundation (30425021, 0637S12442),
Basic Research Foundation (2002CB713807), and Shanghai Key
Project of Basic Science Research (04DZ14005).
Supporting Information Available: Tables listing the
identified unmodified peptides and phosphopeptides; the 134
peptides groups with nonphosphopeptides, monophosphopeptides, and multiphosphopeptides; and the identification
details of all unmodified peptides and phophopeptides. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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