Comprehensive Profiling of Phosphopeptides Based on Anion

Aug 3, 2010 - Song Nie, Jie Dai, Zhi-Bin Ning, Xing-Jun Cao, Quan-Hu Sheng, and Rong Zeng*. Key Laboratory of Systems Biology, Institute of Biochemist...
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Comprehensive Profiling of Phosphopeptides Based on Anion Exchange Followed by Flow-Through Enrichment with Titanium Dioxide (AFET) Song Nie, Jie Dai, Zhi-Bin Ning, Xing-Jun Cao, Quan-Hu Sheng, and Rong Zeng* Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China Received March 30, 2010

For large-scale analysis of phosphorylation at proteome-wide scale, a variety of affinity-based strategies have been developed to enrich phosphopeptide. Because each method differed in their specificity of isolation, the global and unbiased enrichment of phosphopeptides remains a major technical challenge in phosphoproteomics. In the present work, we demostrate that the phosphopeptide enrichment method based on an online continuous pH gradient in a strong anion exchange column (SAX method) is highly complementary to the method based on titanium dioxide (TiO2) affinity enrichment. Moreover, we found that the flow-through fraction of either SAX or SCX is very phosphopeptide-rich, which necessitates further analysis by complementary method. Here, we developed a comprehensive phosphopeptides profiling strategy based on anion exchange followed by flow-through enrichment by TiO2 (AFET). In this strategy, SAX method was used as the first separation/enrichment step, which was online coupled with LC-MS/MS. The phosphopeptides in the SAX flow-through fraction were further enriched with TiO2. As a result, a more comprehensive, less biased phosphoproteome was aquired. Careful comparison of four different combination strategies reveal that the AFET method showed the advantages of more identified phosphopeptides, less mass spectrometry analysis time, as well as simple and automatic process step. It is well-suited for robust and reproducible phosphoproteomics, especially in the case of small amounts of sample. Keywords: Phosphopeptide • anion exchange chromatography • titanium dioxide • phosphoproteomics

Introduction Among the several hundred known posttranslational modifications (PTMs),1 protein phosphorlation is one of the most important PTMs involved in a variety of biological processes such as proliferation, differentiation, and apoptosis in living cells. Characterization of phosphorylation status, and the subsequent decipherment of the relationship between kinases/ phosphatases and their substrates, is crucial to the elucidation of the complex network of signaling and the understanding of disease and drug actions mechanism.2,3 To date, no phosphoproteome has been completely elucidated, but it has been estimated that more than 100000 phosphorylation sites may exist in the human proteome.4 Despite its ubiquitous role, the substoichiometric feature and the high dynamics of phosphoproteins put high demands on comprehensive and specific enrichment of phosphorylated peptides. Recently, many techniques and strategies have been specifically developed to isolate and enrich phosphorylated peptides prior to mass spectometric (MS) analysis, such as the antibody-based method,5,6 chemical derivatization method,7,8 immobilized metal affinity chromatography (IMAC),9-11 hydrophilic interaction chromatography (HILIC),12,13 ion exchange chromatography (SCX/SAX),14-17 * To whom correspondence should be addressed. E-mail: [email protected]. 10.1021/pr100632h

 2010 American Chemical Society

and calcium phosphate precipitation as well.18,19 Different methods provide different results, and each of them has its advantages and disadvantanges.20-23 For instance, the general drawback of chemical derivatization method is the contamination from peptides with other modifications that also undergo chemical derivatization. Furthermore, chemical tagging of nonphosphorylated serine residues has also been detected, and sample may be lost at each step due to the multiple chemical reactions.24 IMAC is one of the most common strategies for phosphopeptide enrichment.9,10 However, nonspecific binding of acidic and nonphosphorylated peptides often hampers successful analysis. To address this problem, Ficarro and colleagues considerably improved the specificity of phosphopeptide enrichment by derivatizating the carboxylic groups on acidic amino acid residues in peptides by O-methylation esterification,25 but this step may introduce unwanted side reactions and loss of peptides due to extensive lyophilization.26 Titanium dioxide (TiO2), as an effective material to isolate phosphopeptides from complex proteolytic digests, has a far higher affinity and better selectivity compared to the IMAC method.27,28 However, owing to the anion-exchange properties of TiO2 at low pH, nonphosphopeptides rich in glutamic and aspartic acid residues might be also retained together with phosphopeptides. To circumvent this problem, 2,5-dihydroxy Journal of Proteome Research 2010, 9, 4585–4594 4585 Published on Web 08/03/2010

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benzoic acid (DHB), glutamic acid, and other acidic reagents29,30 were introduced as a competitive binder in the loading buffers used in TiO2 enrichment of phosphopeptide, which significantly improved the selectivity of TiO2 for phosphopeptide. For large-scale phosphoproteomic studies, single step purification by affinity materials was not effective enough to isolate all phosphopeptides, and strong cation ion exchange chromatography (SCX) is most efficient as a prefractionation technique prior to phosphosphopeptide enrichment using either IMAC or TiO2 chromatography. The method combing SCX with TiO2 (SCX-TiO2 method) has been successfully validated as a powerful and robust combination that was used as the analytical strategy in several large-scale projects.31-33 Recently, strong anion ion exchange chromatography based an online continuous pH gradient (SAX method) was proved to bearobustapprochoflarge-scalephosphoproteomicprofiling.15,34 However, it is no doubted that each phosphopeptide enrichment method has its own bias. To achieve comprehensive phosphoproteome analysis, developing an unbiased phosphopeptides enrichment strategy is necessary. In the present work, we developed a comprehensive phosphopeptides profiling strategy based on anion exchange followed by flow-through enrichment by TiO2 (AFET). In this strategy, SAX method was used as the first separation/enrichment step, which was online coupled with LC-MS/MS. The phosphopeptides in the SAX flow-through faction were further enriched with TiO2. As a result, a more comprehensive, less biased phosphoproteome was aquired.

Materials and Methods Materials. Water was prepared using a Milli-Q system (Millipore, Bedford, MA). Acetonitrile (ACN) and trifluoroacetic acid (TFA) were obtained from Merck (Darmstadt, Germany). Formic acid (FA) was purchased from Aldrich (Milwaukee, WI). Ammonium bicarbonate (NH4HCO3), sodium orthovanadate (Na3VO4), sodium floride (NaF), and glutamic acid was obtained from Sigma-Aldrich Corporation (St. Louis, MO). Urea, dithiothreitol (DTT), CHAPS, and iodoacetamide (IAA) were all purchased from Bio-Rad (Hercules, CA). Fetal bovine serum (FBS) was bought from Gibco Life Technologies (Gibco BRL, Grand Island, NY). Sequencing grade trypsin was obtained from Promega (Madison, WI). All of the chemicals were of analytical purity grade except ACN and FA, which were of HPLC grade. Sample Preparation and Trypsin Digestion. The HeLa cells were cultured in 10 cm plates supplemented with 30 mL of Dulbecco’s modified Eagle’s medium and 10% FBS. When reached 90% confluence, the cells was washed twice with 10 mL of ice-cold PBS. Then, the cells were lysed in the lysis buffer consisting of 8 M urea, 4% CHAPS, 65 mM DTT, 40 mM Tris, 0.2 mM Na3VO4, 1 mM NaF, sonicated, and centrifuged at 25000 g for 1 h. The supernatants were collected and the protein concentration was determined with Bradford assay (Bio-Rad). The tryptic digestion was processed according to the method as described elsewhere.17 The digestion solution was lyophilized and stored at -80 °C for further use. Phosphopeptides Enrichment by a TiO2 Column. Phosphopeptide enrichment by a TiO2 column was carried out as reported by Wu et al.29 Prior to loading sample, the TiO2 column (320 µm × 50 mm, Column Technology, Inc., CA) was equilibrated with 200 µL of loading buffer (65% ACN/2% TFA solution saturated with glutamic acid). Each sample was dissolved with 200 µL of loading buffer and were slowly loaded onto a TiO2 column (3 µL/min) with a syringe pump, the TiO2 4586

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column was sequentially washed with 150 µL of loading buffer, washing buffer I (65% ACN/0.5% TFA), and washing buffer II (65% ACN/0.1% TFA). Then, the bound peptides were eluted with 200 µL of elution buffer I once (300 mM NH4OH/50% ACN) and with 200 µL of elution buffer II twice (500 mM NH4OH/ 60% ACN). Eluates were pooled, lyophilized, and reconstituted in 0.1% FA for MS analysis. Continuous pH Elution SAX-RP-LC-MS/MS Analysis. Orthogonal 2-D LC was performed using a Surveyor liquid chromatography system (ThermoFisher, San Jose, CA). The system comprised a degasser, 2 MS pumps, an autosampler, an SAX column (10 µm, 320 µm × 100 mm, Column Technology Inc., CA), two C18 trap columns (5 µm, 300 µm × 5 mm, Agilent Technologies), an analytical C18 column (RP, 5 µm, 75 µm × 150 mm, Column Technology Inc., CA), and a 15 µm (internal diameter), noncoated SilicaTip PicoTip nanospray emitter (New Objective, Woburn, MA). The continuous pH elution procedure on the SAX column was based on our previous work.15 Briefly, 500 µg tryptic digests were dissolved in 80 µL of pH 7.0 buffer A (5 mM citric acid adjusted by NH4OH) containing 20% acetonitrile, and then loaded into the SAX column. The peptides bound in SAX column were then separated by continuous pH gradient with buffer A (pH 7.0) and buffer B (pH 2.5, 5 mM citric acid adjusted by formic acid), and the last two gradients were formed by mixing buffer B (pH 2.5) with buffer C (pH 2.0, 5 mM citric acid adjusted by formic acid) and maintained at 100% buffer C. The continuous pH gradient (from pH 7.0 to 2.5) was run on the SAX column for 40 h, and every 4 h the SAX column was switched to another RP trap column. Finally, the peptides bound in SAX column were separated into 10 fractions and analyzed by mass spectrometry. The HPLC solvents for reversedphase were 0.1% formic acid (v/v) aqueous (A) and 0.1% formic acid (v/v) in acetonitrile (B). The RP gradient reached 35% mobile phase B in 165 min. The flow rate was 140 µL/min before splitting and 250 nL/min after splitting. SCX-TiO2-RP-LC-MS/MS Analysis. The peptides fractionation by SCX chromatography was conducted according to Beausoleil’s protocal.14 Briefly, 500 µg of HeLa cell protein tryptic digests were redissolved in 20 µL of SCX buffer A (5 mM KH2PO4, pH 2.65/30% acetonitrile). Strong cation exchange chromatography was carried out on a SCX column (320 µm × 100 mm, Column Technology, Inc., CA) at a flow rate of 6 µL/ min. After a 4 min flow-through by buffer A (5 mM KH2PO4, pH 2.65/30% acetonitrile), peptides was eluted with a 32 min linear gradient of 0-21% buffer B (5 mM KH2PO4, pH 2.65/ 30% acetonitrile/350 mM KCl). Finally, 100% buffer B was used to elute all the tightly bound peptides for 4 min. Ten fractions, collected at every 4 min, were lyophilized them for subsequent phosphopeptide enrichment by offline TiO2 chromatography. The enriched phosphopeptide mixtures were ultimately separated by reversed-phase HPLC and MS analysis. The reversedphase HPLC and mass spectrometry conditions were set the same as the continuous pH elution SAX-RP-LC-MS/MS analysis. MS Acquisition. A linear ion trap/Orbitrap (LTQ-Orbitrap) hybrid mass spectrometer (Thermo Fisher, San Jose, CA) equipped with an NSI nanospray source was operated in data dependent mode to automatically switch between in MS and MS/MS acquisition with ion transfer capillary of 200 °C and NSI voltage of 1.85 kV. Normalized collision energy was 35.0%. The mass spectrometer was set as each full MS scan followed by 10 MS/MS scans on the 10 most intense ions from the MS spectrum with the following Dynamic Exclusion settings: repeat

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Profiling of Phosphopeptides Based on AFET

Table 1. Sample Loading Amount, Phosphopeptide Counts, Unique Phosphopeptide Counts, Phosphopeptide Modified Site Counts, MS Run Time, and Phosphopeptide Ratio of Each Method TiO2-only

sample loading amount (mg) phosphopeptide count unique phosphopeptide count modified site count run time (H) ratio of phosphopeptide (%)

0.5 2610 929 920 4 89

SAX

0.5 5516 1516 1638 40 33.4

count 2, repeat duration 0.5 min, exclusion duration 90 s. The resolving power of the Orbitrap mass analyzer was set at 100000 (m/∆m 50% at m/z 400) for the precursor ion scans. Multistage activation (MSA) was enabled in the MS/MS events to improve fragmentation spectra of phosphopeptides.35 All data were recorded with Xcalibur software (Thermo Fisher Scientific, San Jose, CA). DATA Analysis. The acquired MS/MS spectra were searched against the target-decoy database36 consisting of forward and reversed sequences of the proteins in human International Protein Index protein sequence database (IPI human, version 3.28) using the TurboSEQUEST program in the BioWorks 3.2 software package. Database searches were performed by applying the precursor ion m/z tolerances of 50 ppm and fragment ion m/z tolerances of (1 Da. Cysteine residues were statically modified by 57.02146 Da due to carboxyamidomethylation. Dynamic modifications were permitted to allow for the detection of oxidized methoinine residues (+15.99492), and phosphorylated serine, threonine, and tyrosine residues (+79.96633). The maximum number of internal cleavage site was set to 1. For phosphopeptide identification and phosphorylation site localization, all accepted SEQUEST result must have a ∆Cn score of at least 0.1 regardless of charge state, as ∆Cn g 0.1 is significant for discriminating the first candidate peptide from the second candidate peptides. All output results were filtered and combined together using the in-house software named BuildSummary. The peptides and phosphopeptides identified by LTQ-Orbitrap were calculated separately and filtered by precursor ion tolerance m/z e10 ppm and 1.0% false-discovery rate (FDR).37 To eliminate redundancy, if the same peptide(s) were assigned to multiple proteins, the multiple proteins were clustered to a “protein group”. If all of the peptides in protein group A were covered by protein group B which has other peptide assigned, then protein group A was removed. The pI values of the original peptide backbone from all identified phosphopeptides were calculated using an inhouse built Java tool, named BioSolution, where the amino acid pKa’s were used as reported in ref 38.

Results and Discussion The High Complementarity of SAX and TiO2-Based Phosphopeptide Enrichment Methods. First, the performance of SAX and TiO2-based phosphopeptide enrichment/separation methods is carefully evaluated and compared in this work. Phosphopeptides from 500 µg of HeLa cell protein tryptic digests were enriched and analyzed by SAX, TiO2-only, and SCX-TiO2 (SCX followed by TiO2) methods, respectively. Ninehundred and twenty-nine unique phosphopeptides (Table 1) were identified by the TiO2-only enrichment without prefractionation. A significantly increase in unique phosphopeptides identification was observed when SAX method and SCX-TiO2 were employed to enrich phosphopeptides, with 1516 and 1686

SCX-TiO2

AFCT

CTFA

AFET

cACT

0.5 5931 1686 1647 40 68.5

0.5 9170 2644 2706 76 44.0

0.5 10197 2362 2367 76 41.5

0.5 7832 2466 2511 40 42.4

1.0 11090 2705 2690 80 42.5

unique phosphopeptides identified respectively. The overlaps among three different experiments were shown in Figure 1A. Only 154 unique phosphopeptides were identified in all the three experiments. Obviously, there is no single enrichment technique currently which has comprehensive advantages over the others. The overall poor overlaps among these methods clearly explained the three enrichment methods have somewhat bias and detected different, partially overlapping segments of the phosphoproteome. To further investigate the performances of these methods, we calculated the theoretical pI values of the original peptide backbone from all identified phosphopeptides, using an in-house built Java tool, BioSolution, where the amino acid pKa’s reported in ref 38 were used. The distributions of theoretical pI value of phosphopeptides identified in these experiments were plotted in Figure 1B. Acidic phosphopeptides were predominantly identified using SAX method, whereas the pIs of identified phosphopeptides by SCXTiO2 and TiO2-only methods were almost evenly distributed. Interestingly, although 10 SCX fractions were collected before TiO2 chromatographic analysis, the phosphopeptide pI profile using SCX-TiO2 method is similar with that of TiO2-only method. From the above results, it is revealed that the SAX method has bias on the enrichment of acidic phosphopeptides, whereas the methods based on TiO2 or SCX-TiO2 has somewhat bias on the enrichment of basic phosphopeptides. Apart from the pI distributions, the distributions of the ratio of singly and multiply phosphorylated peptides in the three methods were compared, as shown in Figure 1C. Among the total 1516 unique phosphopeptides identified, 59.0%, 30.9%, and 10.1% of them were singly, doubly, and triply phosphorylated peptide, respectively. However, the percentages of singly, doubly, and triply phosphorylated peptides in SCX-TiO2 method were 78.8%, 16.2%, and 5.0%, respectively. In the TiO2only enrichment method, 87.7% of total identified phosphopeptide was singly phosphorylated peptides, only 12.3% multiply phosphorylated peptides could be detected. Fractionation by SCX in SCX-TiO2 method did not significantly increase multiply phosphorylated peptides identification. Among those methods, the SAX method showed its potential to detect more multiply phosphorylated peptides, while the methods based on SCX-TiO2 or TiO2 have bias toward the enrichment of more singly phosphorylated peptides. From the above analyses, it is demonstrated that SAX method has a bias to enrich acidic and multiply phosphorylated peptides, and the method based on titanium dioxide (TiO2) affinity enrichment has a tendency to enrich more basic and singly phosphorylated peptides. Thus, SAX method is highly complementary to TiO2-based phosphopeptide enrichment method, which indicates that the combination of these two Journal of Proteome Research • Vol. 9, No. 9, 2010 4587

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Figure 1. (A) Phosphopeptides overlap among TiO2-only, SAX, SCX-TiO2 methods. (B) Distribution of theoretical pI range of peptides found with phosphorylation by SAX, SCX-TiO2, and TiO2-only methods, respectively. (C) Percentage of phosphopeptides with different number of phosphorylated sites in SAX, SCX-TiO2, or TiO2 methods for HeLa cells.

complementary methods would provide a more comprehensive phosphoproteome. The Flow-Through Fraction of Either SCX or SAX Need to Be Further Analyzed. For complex peptide mixtures, a single step purification by affinity materials is not effective enough to isolate all phosphopeptides. Ion exchange chromatography isapowerfulapproachtoseparateandenrichphosphopeptides,9,14,33,34,39,40 but there is no much attention paid to the analysis of the flow4588

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through fraction of either SCX or SAX. The following results show that the flow-through fraction of either SCX or SAX is very phosphopeptide-rich, which necessitates further analysis. In this work, 500 µg tryptic digests from HeLa cells were separated into 10 fractions by SCX, each fraction was enriched by TiO2, then analyzed by one-dimensional LC-MS/MS method. The distribution of phosphopeptides identified in each fraction of SCX separation was shown in Figure S1 of Supporting

Profiling of Phosphopeptides Based on AFET Information. The flow-through fraction of SCX separation identified much more phosphopeptides than others, which indicated the flow-through fraction is very phosphopeptide rich. To comprehensive profile phosphoproteome, the flowthrough fraction was only analyzed several times by TiO2 enrichment in some laboratories,33,41 but it is not sufficient. To date, there are no further deeply investigations on the flowthrough fraction. Nevertheless, in the SAX method, only the bound fraction was online analyzed, the flow-through fraction was not further analyzed.15 In this work, more than 1000 unique phosphopeptides were identified in the flow-through fraction of SAX by TiO2 enrichment. Obviously, the flow-through fraction of either SCX or SAX is very phosphopeptide-rich. Ion exchange chromatography separate peptides based primarily on ionic charge.42,43 Therefore, many of phosphopeptides can not be remained in the SCX and SAX chromatography column due to their charge state. At pH 2.7, Gygi and colleagues predicted that 68% of in silico tryptic peptides in the human database have a net charge of 2+, any of these peptides would still have a net charge state of 1+ after the addition of a single phosphate,14 these peptides would bind to SCX column when loaded with pH 2.7 loading buffer. Nevertheless, the remaining peptides with net charge state less than 2 and multiply phosphorylated peptides would not retain on the SCX column under this condition due to their net zero or negative charges. Therefore, the flow-through fraction should be rich in phosphopeptides, especially multiply phosphorylated peptides. Similarly, at pH 8.5, 29.2% in silico tryptic peptides have positive net charge between 0 and +2.15 In the process of sample loading, many singly phosphorylated peptides and basic phosphorylated peptides will not be retained on the SAX column under these conditions owing to their net zero or positive charges. Therefore, the flow-through fraction of either SCX or SAX is very phosphopeptide-rich, which necessitates further analysis by complementary method. The Phosphopeptides in Unbound Fraction of SCX/SAX Chromatography. As proven above, SAX method has the merits of high-degree complementation with SCX-TiO2 method in phosphopeptide enrichment, as well as the ability of separation/enrichment of multiply phosphorylated peptides. Thus, SAX method was employed to analyze phosphopeptides in the flow-through fraction (unbound) of the SCX. Similarly, SCXTiO2 method was employed to analyze phosphopeptides in the flow-through fraction (unbound) of the SAX. In total, 1356 unique phosphopeptides were identified in unbound fraction of SCX by SAX method, and 1222 unique phosphopeptides were identified in the unbound fractions of SAX by SCX-TiO2 method. These results strongly supported that the unbound (flow-through) fraction of SCX/SAX separation was phosphopeptide-rich and needed to be further separated and enriched by complementary method to obtain a complete view of phosphoproteome of whole organelle/cell. More than 1200 unique phosphopeptides were identified in both the unbound fraction of SCX and SAX, but the chemical feature of phosphopeptides in the unbound fraction of SCX/ SAX is significantly different because SCX and SAX chromatography separate peptides based primarily on ionic charge.42,43 Acidic and multiply phosphorylated peptides dominated in the unbound fraction of SCX, whereas more basic and singly phosphorylated peptides were contained in the unbound fraction of SAX. The distributions of theoretical pI value of identified phosphopeptides in the unbound fraction are plotted in Figure 2A. It is obvious that more acidic peptides were

research articles detected in the unbound fraction of SCX, while more basic peptides were identified in the unbound fraction of SAX. On the other hand, the ratios of multiphosphopeptides versus total phosphopeptides identified in each unbound fraction were also different, as shown in Figure 2B. Among the total 1356 unique phosphopeptides identified in the unbound fraction of SCX, 58.9%, 31.4%, and 9.7% of them were singly, doubly, and triply phosphorylated, respectively. However, the percentages of singly, doubly, and triply phosphorylated peptides in the unbound fraction of SAX were 79.0%, 18.3%, and 2.7%, respectively. To further investigate the nature of phosphopeptides in flowthrough fraction of SCX, the pI distribution of phosphopeptide of bound fraction analyzed by SCX-TiO2 method and unbound fraction analyzed by SAX method were plotted and shown in Figure S2 of Supporting Information. A large number of acidic peptides were identified in the unbound fraction by SAX method. Among 1356 unique phosphopeptides identified in unbound fraction by SAX method, 425 were doubly phosphorylated peptides and 128 were triply phosphorylated peptides. However, only 158 doubly phosphorylated peptides and 18 triply phosphorylated peptides were identified in the flowthrough fraction by TiO2 enrichment. More than 3 times of the number of multiphosphopeptides was obtained in the analysis of flow-through fraction by SAX method. Significant increase of multiphosphopeptides identification in the unbound fraction of SCX agrees well with the nature of SCX and SAX. As a result, the flow-through fraction of SCX analyzed by SAX method is necessary because acidic and multiply phosphorylated peptides dominated in the unbound fraction of SCX. In the unbound fractions of SAX, 1007 and 1222 unique phosphopeptides were identified by SCX-TiO2 and TiO2-only method, respectively. Although 10 fractions were analyzed in SCX-TiO2 method, the number of identified phosphopeptides did not significantly increase. The phosphopeptides identified by the two methods presented identical theoretical pI profile and ratio of multiphosphopeptide. However, the whole analysis process of SCX-TiO2 method is carried out off-line, which is very time-consuming and labor intensive. Therefore, the flowthrough fraction of SAX analyzed by TiO2-only method is feasible because most monophosphopeptides and basic phosphopeptides existed in the unbound fraction of SAX. SAX Chromatography Has Better Resolution in Phosphopeptide Fractionation. In this study, the cross-overlap of SAX and SCX were compared. Among 10 successive elution fractions of SAX (Table S1, Supporting Information), 78.57% of phosphopeptides were detected in only a single fraction, 12.08% were detected in two fractions, 4.48% were detected in three fractions, 2.24% were detected in four fractions, and only 2.62% were detected only in over four fractions. In contrast, 68.01%, 17.40%, 7.93%, 3.04%, and 3.61% of phosphopeptides were detected in one, two, three, four, and over four fractions of SCX in SCX-TiO2 method, respectively. By comparsion above, it was found that the SAX in the phosphopeptide separation had better resolution, namely lower cross-overlap than SCX. A Comprehensive Phosphopeptides Enrichment Strategy Based on Anion Exchange Followed by Flow-Through Enrichment with Titanium Dioxide (AFET). On the basis of the complementary features of the SAX and TiO2 methods, we developed a comprehensive phosphopeptides profiling strategy, anion exchange followed by flow-through enrichment by TiO2 (AFET). In this strategy, SAX is used as the first separation/ enrichment step, which is online coupled with a mass specJournal of Proteome Research • Vol. 9, No. 9, 2010 4589

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Figure 2. (A) Distribution of theoretical pI range of peptides found with phosphorylation in the unbound fractions of SCX and SAX. (B) Percentage of phosphopeptides with different number of phosphorylated sites in the unbound fractions of SCX and SAX for HeLa cells.

trometer. The flow-through faction was further enriched with TiO2. As a result, a more comprehensive, less biased phosphoproteome could be acquired. The whole procedure of phosphopeptide enrichment/separation was depicted in Figure 3A. Then 500 µg tryptic digests from HeLa cells were analyzed by the AFET method. In total, 2466 unique phosphopeptides and 2511 phosphorylation sites were identified (Table 1). Figure 4 shows the overlap between the bound fraction and the flowthrough fraction (unbound). Only 46 unique phosphopeptides were identified in both fractions, which revealed that all phosphopeptides were effectively separated into two fractions of bound and unbound fraction by SAX column. The theoretic pI distribution of phosphopeptides from bound and unbound fractions is shown in Figure 5A. Acidic and multiply phosphorylated peptides were bound on SAX column, and basic and 4590

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singly phosphorylated peptides were retained in flow-through fraction. From the above analysis, we can confirm the AFET combination strategy is highly complementary phosphopeptide enrichment strategy, which could be beneficial to the comprehensive phosphoproteome analysis. Moreover, the human IPI database was digested in silico by using an in-house built Java tool. To simplify the data, no missed cleavage was allowed in the in silico tryptic digestion, then the pI values were calculated. The pI distributions of phosphopeptides identified by several combination strategies and the in silico tryptic peptides from human IPI database are shown in Figure 5B. The pI profiles observed using SCX-TiO2 method pleasingly resembled the profile of an in silico tryptic peptides of the complete human protein database, but it cannot represent that the SCX-TiO2 method is a comprehensive phosphopeptide enrichment method

Profiling of Phosphopeptides Based on AFET

Figure 3. Experiment workflows of different combination strategies. (A) Experiment workflow of AFET method. The tryptic peptides were efficiently separated into two fractions by SAX column, of which the phosphopeptides of the bound fraction on SAX column was fully automatically online enriched and analyzed by SAX method, and the phosphopeptides of the flowthrough fraction was enriched using TiO2 and analyzed by LCMS/MS. (B) Experiment workflow of AFCT, CTFA, cACT methods.

Figure 4. Overlap of unique phosphopeptides identified by SAX method (bound fraction) and TiO2 method (flow-through fraction) in AFET method.

and allowed for complete analysis of the phosphoproteomic analysis because it is obvious that a large part of phosphopeptides in the flow-through fraction is neglected in the method. The pI profiles observed using AFET method is obviously different to the profile of an in silico tryptic peptides of the complete human protein database, with a higher percentage at the acidic range and a similar profile at the basic range. To date, no phosphoproteome has been completely mapped, so we do not have a standard for benchmarking, but we have presented a method that did not biased against either acidic or basic phosphopeptides compared with phosphopeptide enrichment method based on TiO2. Comparison of AFET with Other Methods. In the current work, we further compared AFET method with other three different combinational strategies. The whole procedure of phosphopeptide enrichment/separation was depicted in Figure 3B. The first combinational method is based on SCX-TiO2, followed by flow-through enrichment by SAX method (CTFA method). Then 500 µg tryptic digests from HeLa cells were

research articles separated individually into 10 fractions by SCX, the flowthrough fraction was loaded on a SAX column, online separated into 10 fractions, and analyzed by MS. The other nine fractions were processed to enrich phosphopeptides by a TiO2 column and MS analysis. We termed this strategy as a CTFA method. The second combinational method is based on anion exchange followed by flow-through enrichment by SCX-TiO2 (AFCT method); 500 µg tryptic digests from HeLa cells were analyzed. The flow-through fraction of SAX was lyophilized and dissolved into pH 2.7 SCX loading buffer, and those fractions were further analyzed using SCX-TiO2 method. The bound peptides on SAX were online analyzed by SAX method. We named this strategy as AFCT method. Third, except the AFCT and CTFA methods described as above, another combinational strategy was based on the combination of independent SAX and SCX-TiO2 method (cACT method). In the experiment of the cACT method, 500 µg tryptic digests from HeLa cells were loaded in SAX and SCX, respectively. SAX and SCX-TiO2 methods were performed individually, and the results were combined. The detail identification results of phosphopeptides are listed in Table S1 of Supporting Information. The amounts of sample loading, the counts of phosphopeptide, the counts of unique phosphopeptide, the counts of phosphopeptide modification sites, MS run time, and the ratio of phosphopeptides of each method are summarized in Table 1. Among the four combination strategies, AFCT method identified the highest number of unique phosphopeptides and phosphorylated modification sites. The CTFA method was relatively inferior to the other three other combinations. Although the amount of sample loading was doubled in the methods of cACT method, only a few counts of phosphopeptides were increased. However, the MS analysis time in the AFET method is half of the others, and there was no significant decrease about the number of identified unique phosphopeptides and phosphorylated modified sites. If equal MS time was used, the two replicated experiments of AFET combination strategy identified more unique phosphopeptides than other combination strategies, involving 3426 unique phosphopeptides, 3485 unique phosphorylated sites, and 14778 phosphopeptides counts. Moreover, in AFET strategy, all the phosphopeptides bound on SAX column is online analyzed by mass spectrometry and the unbound fraction is analyzed by TiO2 method without prefractionation. About the enrichment efficiency, there are no significant different in the ratio of phosphopeptides detected in each combination methods, and more than 40% phosphopeptides was achieved in all these combination methods. To evaluate the experimental variation, each combination method was replicated. The average unique phosphopeptides and variation in the replicated experiments ae plotted in the bar graph (Figure 6). From the picture, we could find that the variation of CTFA method is the most and that of other three methods were almost identical. The results reveal AFET method has good reproducibility. Thus, by comparing with these methods, AFET method has the advantage of high automation, of less MS analysis time-consuming, and of more simple process step. Recently, some new methods have been developed to improve phosphoproteome analysis. The works of Ishihama et al.,44 Heck et al.,45,46 and Larsen et al.26 are summarized in Table S2 of Supporting Information in detail. Submilligram levels of material were used in these sensitive analyses, such as Heck et al. analyzing more than 2100 unique phosphopeptides in a HeLa cell sample using the SCX-TiO2 method. However, SCX-TiO2 method used in the Heck’s experiment is Journal of Proteome Research • Vol. 9, No. 9, 2010 4591

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Figure 5. (A) Distribution of theoretical pI range of peptides found with phosphorylation by SAX (bound fraction) and TiO2 methods (flow-through fraction) in AFET method. (B) Profile of theoretical pI range of peptides found with phosphorylation by SCX-TiO2, AFCT, AFET methods, and of in silico tryptic peptides digested from proteins in human IPI database.

Figure 6. Average unique phosphopeptides and variation in the replicated experiments.

the same as our SCX-TiO2 method, which has bias toward the enrichment of monophosphopeptides and basic phosphopep4592

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tides, which is proved by our results. The same bias was found in the experiment by analyzing the data. Among 1800 unique phosphopeptides obtained from HeLa cell using successive elution TiO2 method in Ishihama group, 76.1%, 19.6%, and 4.2% of them was singly, doubly, and triply phosphorylated, respectively. The profile of phosphopeptides is identical to our results by only the TiO2-only and SCX-TiO2 methods, and successive elution could not increase the identification of multiphosphopeptide. Moreover, more phosphopeptides were identified by our AFET method compared with these methods. IMAC also tends to enrich multiply phosphorylated peptides, much like the SAX approach, but the ratio of multiphosphopeptides is only 37.8% in the SIMAC method, lower than 42% in the AFET method employed in our experiment. A 120 µg sample was used in the SIMAC analysis, and the number of identified unique phosphopeptides is far lower than that of our identified phosphopeptides by the AFET method (492 vs 2466), let alone

research articles

Profiling of Phosphopeptides Based on AFET 26

the generous 6.0% FDR criterion in this report. On the other hand, the whole process of SIMAC analysis is conducted offline, which is very time-consuming and labor intensive. Thus, compared with these methods, AFET method can provide a more comprehensive phosphoproteome and has the advantage of high automation despite more protein sample used. In this report, traditional TiO2 column is used to enrich phosphopeptide and its bias toward monophosphopeptides has been proved by our experiments and other work.23 However, when TiO2 beads were used, Li and Ning47 found that deficient beads can be helpful to identify much more multiply phosphorylated peptides. Use of Differential Enrichment Strategies According to Sample Amounts. Differential enrichment strategies could be chosen according to the total sample amount available to maximize the phosphopeptide identifications efficiently. On the basis of this work, the AFET method was a better choice if limited sample amount is available. In AFET strategy, all the phosphopeptides bound on SAX column is online analyzed by mass spectrometry and the unbound fraction is analyzed by TiO2 method without prefractionation, which is operated offline and easily leads to sample loss.48 Moreover, more than 2500 unique phosphopeptides were identified using AFET strategy when 500 µg tryptic digests were used in this study. The profile of phosphopeptides identified in the AFCT strategy is the identical to that in the AFET strategy (Figure 5B). If the sample amount is enough, we recommend AFCT strategy to maximize the phosphopeptides identification. Enrichment of phosphopeptides is prerequisite for large amount of sample to profile global phosphoproteome. When large amounts of sample were loaded on a SAX column, this flow-through fraction is very phosphopeptide rich and is not sufficient to analyze several times by TiO2 enrichment. Therefore, it is necessary that the flow-through fraction is analyzed by the SCX-TiO2 method. In conclusion, the phosphopeptides enrichment strategy is very flexible and different combinations could be chosen in different situations and purposes.

Conclusion In this work, we presented a comprehensive phosphopeptides enrichment strategy that has little bias to either acidic or basic phosphopeptides. It is a novel system using SAX method as a first separation/enrichment step, followed by flow-through enrichment with titanium dioxide, which achieved robust separation and detection of phosphopeptides in complex cell proteome. The robust phosphopeptides enrichment strategy can be flexibly applied in different situations. The use of multiple proteolytic reagents and novel peptide dissociation mode (ETD) in combination with the phosphopeptide enrichment strategy will ultimately result in high recovery levels of the full phosphoproteome.

Acknowledgment. This work was supported by the National Natural Science Foundation (30425021, 30521005), Basic Research Foundation (2006CB910700), CAS Project (KSCX2-YW-R-106), High-Technology Project (2007AA02Z334), and Proteomage Project of EU. Supporting Information Available: Cross-overlap of identified phosphopeptides among 10 successive elution fractions of SAX and SCX (SCX-TiO2 method). Comparison of this work with other works from recently published papers. Unique phosphopeptides Identified from HeLa cell protein sample by

different enrichment strategies in this study. Distribution of unique phosphopeptides in each fraction of SCX-TiO2 method. Distribution of theoretical pI range of phosphopeptides in bound and unbound fractions of SCX analyzed by SCX-TiO2 and SAX methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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