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†Freiburg Institute for Advanced Studies (FRIAS), School of Life ... Department of Dermatology, University Freiburg Medical Center, Hauptstrasse 7, ...
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Combinatorial Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) and Strong Cation Exchange (SCX) Chromatography 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 and ∥BIOSS Centre for Biological Signalling Studies, University of Freiburg, Albertstrasse 19, 79104 Freiburg, Germany ‡ ZBSA Center for Biological Systems Analysis, University of Freiburg, Habsburgerstrasse 49, 79104 Freiburg, Germany § Department of Dermatology, University Freiburg Medical Center, Hauptstrasse 7, 79104 Freiburg, Germany S Supporting Information *

ABSTRACT: In large-scale phosphoproteomics studies, fractionation by strong cation exchange (SCX) or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is commonly used to reduce sample complexity, fractionate phosphopeptides from their unmodified counterparts, and increase the dynamic range for phosphopeptide identification. However, these procedures do not succeed to separate, both singly and multiply phosphorylated peptides due to their inverse physicochemical characteristics. Hence, depending on the chosen method only one of the two peptide classes can be efficiently separated. Here, we present a novel strategy based on the combinatorial separation of singly and multiply phosphorylated peptides by SCX and ERLIC for in-depth phosphoproteome analysis. In SCX, mostly singly phosphorylated peptides are retained and fractionated while not-retained multiply phosphorylated peptides are fractionated in a subsequent ERLIC approach (SCX− ERLIC). In ERLIC, multiply phosphorylated peptides are fractionated, while not-retained singly phosphorylated peptides are separated by SCX (ERLIC−SCX). Compared to single step fractionations by SCX, the combinatorial strategies, SCX−ERLIC and ERLIC−SCX, yield up to 48% more phosphopeptide identifications as well as a strong increase in the number of detected multiphosphorylated peptides. Phosphopeptides identified in two subsequent, complementary fractionations had little overlap (5%) indicating that ERLIC and SCX are orthogonal methods ideally suited for in-depth phosphoproteome studies. KEYWORDS: ERLIC, SCX, MDLC, phosphoproteomics, TiO2, mass spectrometry

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INTRODUCTION

ERLIC and SCX barely retain singly and multiply phosphorylated peptides, respectively.12,16 In SCX, under acidic conditions (pH < 3), more than 68% of tryptic peptides have a net charge of +2 which would drop to +1 after the addition of a phosphate group. These peptides can still be retained on a SCX column. However, the remaining phosphopeptides with net charge states of zero or below will not bind to the column and elute in the flow through (FT).16,17 Consequently, most multiphosphorylated peptides (≥3) elute in the FT and are not fractionated, and hence, most identified phosphopeptides are singly phosphorylated.18 In ERLIC, the phosphate groups of respective peptides are partially ionized and electrostatically retained by the stationary phase. Generally, multiphosphorylated peptides bind more strongly to the stationary phase due to the higher number of negative charges deriving from the phosphate groups.19 In contrast, singly and nonphosphorylated peptides elute in the FT. Therefore, the

Thirty percent of all proteins within a cell are potentially phosphorylated and may participate in signaling pathways and regulation of cellular functions at a given time point.1,2 Hence, phosphorylation dynamics have been studied in various biological settings.3−6 Because of the low abundance of specific phosphosites within proteins and the chemical heterogeneity of respective phosphopeptides, fractionation and enrichment steps prior to reversed phase (RP)-liquid chromatography−mass spectrometry (LC−MS) analysis of peptides are crucial.4,7,8 Thus, for large-scale phosphoproteomic studies, two-dimensional chromatography has become a method of choice to reduce sample complexity and enrich phosphorylated peptides.9−11 The combinations of offline strong cation exchange (SCX) or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with RP chromatography (SCX-RP or ERLIC-RP) are most widely used.12−15 Both methods have advantages and disadvantages: ERLIC and SCX provide good orthogonality to RP chromatography prior to MS analysis, but © 2012 American Chemical Society

Received: April 20, 2012 Published: July 7, 2012 4269

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and deoxycholate were removed by centrifugation. The protein concentration was measured using the bicinchoninic acid assay (Thermo Fisher Scientific, Bonn, Germany) and tryptic digests were stored at −80 °C for further use.

FTs of both SCX and ERLIC are very complex necessitating further fractionations by complementary methods. In most global phosphoproteomics studies, FTs are introduced to three consecutive phosphopeptide enrichments by TiO2 or IMAC which are commonly not sufficient to unravel the underlying complexities.18,20−22 To overcome this problem, multidimensional (or combinatorial) liquid chromatography (MDLC) approaches are urgently needed which generally aim to reduce sample complexity and improve resolving power and peak capacity.23 Thus, it could be shown that three-dimensional on-line MDLC can significantly improve identification and quantification of iTRAQ-labeled peptides compared to traditional methods. In the respective example, a SCX column was used as trap column between two RP columns (RP-SCX-RP).24 Recently, a similar study showed that offline SCX fractionation coupled to on-line RP−RP (high- and low-pH) can increase the quality of protein identification and quantification of iTRAQ-labeled chloroplast proteins.25 The benefits of additional separation dimensions as compared to a long gradient or multiple replicate runs for in-depth phosphoproteomics analysis were also demonstrated recently. Weak anion exchange (WAX) was employed as a complementary fractionation method with traditional SCX. An increase of 40% in the number of unique phosphopeptides was achieved compared to a one-dimensional fractionation.16 Although both, ERLIC and SCX, are powerful methods for fractionation of phosphopeptides, so far, not much attention has been paid to their combinatorial use in phosphopeptide analyses.21 In the current study, we investigate the fractionation performance of SCX and ERLIC for combinatorial reseparation of respective FTs as compared to the common one-dimensional SCX fractionation of phosphopeptides. The FT of the first dimension (SCX or ERLIC) was reseparated using the complementary method to harness advantages of both approaches. The efficiency of each combination was evaluated based on the number of detected phosphopeptides, percentages of singly and multiply phosphorylated peptides, as well as their distribution over the gradients.

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SCX-Only Workflow

SCX chromatographic separations were performed on an Akta System using a 1 mL column (Resourse S, GE Healthcare) with a 1 mL loop. Tryptic digest of 6 mg HeLa proteins were acidified by FA to a pH under 3, diluted to a final concentration of 30% ACN, and loaded to an equilibrated column with 30% ACN containing 5 mM KH2PO4, pH 2.7 (Solvent A). After 5 min at 100% A, peptides were eluted with 30% ACN containing 5 mM KH2PO4 and 300 mM KCl, pH 2.7 (Solvent B) using a gradient from 0% to 35% in 30 min followed by 35−100% B in 5 min and then maintained at 100% B for 10 min at a flow rate of 1 mL/min. SCX−ERLIC Workflow

Similar conditions as described above with minor changes in the gradient slope were used for SCX. After injection, the column was kept at 100% A for 5 min, peptides were eluted with 30% Solvent B using a gradient from 0% to 30% in 28 min followed by 30% to 100% B in 3 min, and then maintained at 100% B for 10 min. Collected FT was diluted by ACN to a final concentration of 70% and loaded by a 10 mL loop to an equilibrated ERLIC column. ERLIC separation was performed on an Akta System using an in-house packed Poly-WAX LP column (5 μm particle size, 300 Å pore size; PolyLC, Columbia, MD; Tricorn 5/100; GE Healthcare). The gradient was created using a combination of 10 mM sodium methylphosphonate (Na-MePO4) with 70% ACN, pH 2.0 (Solvent A), and 200 mM triethylamine phosphate (TEAP) with 60% ACN, pH 2.0 (Solvent B). Thirty-five minutes at 100% A was followed by a ramp from 0% to 25% B for 3 min and 25−100% B for 25 min and then maintained at 100% B for 10 min at a flow rate of 0.5 mL/min. ERLIC−SCX Workflow

Similar conditions as described above with minor changes in the gradient slope were used for ERLIC. Samples were diluted by ACN to a final concentration of 70% and loaded by 2 mL loop to an equilibrated ERLIC column. Thirty-four minutes at 100% A was followed by a ramp from 0% to 25% B in 3 min and 25−100% B in 20 min, and, finally, 100% B for 10 min at a flow rate of 0.5 mL/min. ERLIC-FT was concentrated to reduce the ACN concentration to 30%, acidified with FA, and loaded to an equilibrated SCX column by a 10 mL loop. After 17 min at 100% A, peptides were eluted using a gradient from 0% to 30% B in 30 min followed by 30−100% B in 5 min, and then maintained at 100% B for 10 min at a flow rate of 1 mL/min.

EXPERIMENTAL METHODS

Chemicals

All solvents, acetonitrile (ACN) (Wako, Neuss, Germany), acetic acid (LGC Promochem, Wesel, Germany), and formic acid acid (FA) (Merck, Darmstadt, Germany) were LC−MS grade. Sodium deoxycholate, dithiothreitol (DTT), ammonium bicarbonate (ABC), iodoacetamide (IAA), dihydroxybenzoic acid (DHB), and triethylamine (TEA) were all purchased from Sigma Aldrich (Munich, Germany). Potasium 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).

TiO2 Enrichment

Ten microliters of a 30% TiO2 slurry (MZ-Analysentechnik, Mainz, Germany) in 30 mg/mL DHB (Sigma)26 was added to each fraction and FT and incubated for 30 min at room temperature. Beads were washed with 100 μL of 10% ACN and 1% TFA followed by 100 μL of 80% ACN and 1% TFA and finally 100 μL of water. Phosphopeptides were eluted using 25% ammonium hydroxide in 20% and 40% ACN, respectively. Eluted phosphopeptides were dried to less than 5 μL and resuspended in 15 μL of 0.5% acetic acid for analysis. Five microliters of each fraction was used for MS analysis. For consecutive incubations, the peptide−beads slurry was

Sample Preparation

HeLa cells were grown to near confluence in 15-cm dishes, scraped, and lysed in 1% sodium deoxycholate in 50 mM ABC buffer and the lysate was clarified by centrifugation. Supernatants were reduced with 1 mM DTT and alkylated with 5.5 mM IAA for 45 min at room temperature in darkness before digestion with trypsin (1:75 trypsin/protein) overnight at 37 °C. Digests were acidified to 1% FA and undigested proteins 4270

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Figure 1. Experimental workflow. Proteins in whole cell lysate were digested using trypsin. Peptides were fractionated by ERLIC and SCX, respectively. In SCX-only (bottom workflow), 26 fractions and FT were collected. In the two other cases, FT of the first dimension, ERLIC or SCX, were refractionated using SCX or ERLIC in the second dimension, respectively. Both approaches yielded again 26 fractions, 15 from the first, 11 from the second dimension, and one FT. In all cases, fractions and FT were subjected to TiO2-based phosphopeptide enrichment, one time for fractions, and three times for FT, respectively, and eluates analyzed by RP-LC−MS/MS.

Table 1. Summary of identified Phosphopeptides in Respective Workflows nonredundant phosphopeptides (median score)

a

workflow

phosphopeptides

total

SCX-only SCXa SCX−ERLIC ERLIC−SCX

10666 13585 10831 13486

4373 4923 5300 6433

1st dimension 4373 4632 3454 3345

(77) (85) (80) (81)

2nd dimension

no. of fractions

pSer (%)

pThr (%)

pTyr (%)

706 (70) 2137 (83) 3445 (82)

27 35 27 27

91.1 90.8 90.1 90.1

8.3 8.4 9.4 9.6

0.6 0.8 0.5 0.3

:SCX-FT after repetitive TiO2 incubations subjected to additional ERLIC separation.

a minimum length of six amino acids were used for peptide identification.29 The “Evidence” table was basis for peptide analyses. For nonredundant hits (different peptide sequence or same sequence but different localization of phosphosites), those peptide variants were selected that had the highest localization probabilities for specific sites. No cutoff was applied.

centrifuged and the supernatant was incubated with another aliquot of freshly prepared TiO2 beads. Mass Spectrometric Analysis

Mass spectrometric measurements were performed on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled with an Agilent 1200 nanoflowHPLC (Agilent Technologies GmbH, Waldbronn, Germany) essentially as described.27 HPLC-column tips (fused silica) with 75 μm inner diameter (New Objective, Woburn, MA) were self-packed15 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] with increasing organic proportion was used for peptide separation (loading of sample with 2% B; separation ramp: from 10% to 50% B within 23 min). The flow rate was 250 nL/min and for sample application 500 nL/min. 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 five 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. For MS/MS, wideband activation and multi stage activation were enabled with the neutral loss mass list of singly, doubly, and triply phosphorylated peptides. The mass range for MS was m/z = 350−2000 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 flow; ion-transfer tube temperature 200 °C.

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RESULTS AND DISCUSSION

Experimental Design and Data Analysis

To evaluate the advantages of FT reseparations by complementary chromatographic methods for phosphopeptide identifications by MS-based proteomics, we designed two workflows SCX−ERLIC and ERLIC−SCX which we compared to SCXonly. HeLa cells were lysed using sodium deoxycholate as described previously.18 Six milligrams of HeLa protein digest was subjected to each of the three different workflows (Figure 1). In all approaches, peptides were separated using a FPLC system and resulting fractions were incubated with TiO2 beads for phosphopeptide enrichment. In the case of MDLC separations, 15 fractions and FT were collected in the first dimension. Afterward, the FT was reseparated by the complementary chromatography method (second dimension) after adjusting pH and ACN percentage. Eleven fractions and FT were collected giving rise to a total of 27 fractions. In the case of SCX-only, a longer gradient resulting in 26 fractions of lower volume and FT was used. After phosphopeptide enrichment, each fraction was analyzed by LC−MS/MS using collision induced dissociation and multistage activation as fragmentation technique. Sixty-two min runs (30 min gradients) gave rise to a total MS measurement time of 30 h/experiment. Raw files were analyzed using MaxQuant.28 All workflows were performed minimally by two biological replicates. Identified phosphopeptides, and distribution of peak intensities between different fractions are shown as Supporting Information.

Data Analysis

All raw files were analyzed with the software MaxQuant (Version 1.0.1.18).28 Cysteine carbamidomethylation was selected as fixed modification; methionine oxidation, protein N-terminal acetylation, and phosphorylation on serine, threonine and tyrosine were selected as variable modifications. Up to three miscleavages were allowed. Precursor ion mass tolerance was 7 ppm, and fragment ion mass tolerance was 0.5 Da for MS/MS spectra. A false discovery rate (FDR) of 1% and

Distribution of Phosphopeptides Fractionated by SCX

In our previous study, we showed that SCX yields a higher number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated pep4271

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Figure 2. ERLIC fractionation of SCX-FT after incubation with TiO2 beads leads to additional identifications of multiphosphorylated peptides. (A) Numbers of identified phosphopeptides and their distribution over the ERLIC gradient are shown. Phosphopeptides are colored according to bound phosphate groups (1p to ≥4p). Percentages of nonredundant, identified mono- and multiphosphorylated peptides are depicted in the right corner. (B) Venn diagram of nonredundant phosphopeptides identified in both approaches, analysis of SCX-FT and ERLIC fractions. (C) Venn diagram of singly phosphorylated peptides identified in both approaches. (D) Venn diagram of multiply phosphorylated peptides identified in both approaches.

tides.18 To investigate the advantages and/or limitations of an additional complementary dimension of fractionation in the analysis of complex samples like whole cell lysates, we compared single step fractionation by SCX with two-dimensional fractionation workflows SCX−ERLIC and ERLIC−SCX, as illustrated in Figure 1. A total number of 10 666 phosphopeptides were identified from 27 fractions (26 fractions + FT) by SCX-only, of which 4373 were nonredundant, meaning peptides of different sequence or the same peptides with different phosphorylation sites (Table 1; Supporting Information Table S1). The distribution of nonredundant singly, doubly, and multiply phosphorylated peptides from two biological replicates was 59.3%, 32.3%, and 8.4%, respectively (Supporting Information Figure S1). A major drawback of SCX fractionation is the low identification rate of multiphosphorylated peptides. The majority of these elute in the FT due to repulsion of negatively charged phosphate groups from the SCX stationary phase. Additionally, acidic peptides containing glutamic and asparatic acid residues also elute in the FT and may suppress signals of multiphosphorylated peptides in MS analyses. Therefore, most phosphoproteomics studies incubate FTs three consecutive times with TiO2 beads to unravel the complexity of contained multiphosphorylated peptides. We followed this protocol and identified 2951 phosphopeptides in nine LC−MS analyses, three technical replicates of three TiO2 beads incubations. A total of 968 of these were nonredundant. We reseparated this specific FT by ERLIC to explore the number and composition of phosphopeptides still contained in the FT after three consecutive TiO2 incubations. We used a fast slope gradient, collected 8 fractions (7 fractions and FT), subjected these to TiO2 incubations, in the case of FT three consecutive rounds, and analyzed eluted peptides by LC−MS/MS. The percentages

of identified, nonredundant mono- and multiphosphorylated peptides in each fraction are depicted in Figure 2A. A total of 1103 phosphopeptides were identified, of which 706 were nonredundant (Table 1; Supporting Information Table S1). About 50% of the detected phosphopeptides were also identified in the SCX-FT (Figure 2B). But a majority of identified multiphosphorylated peptides (more than 74%) were new as compared to SCX (Figure 2C,D). As previously shown, multiphosphorylated peptides have a higher affinity to TiO2 and should be enriched preferentially. However, as we identified 218 new multiphosphorylated peptides after ERLIC fractionation, it appears that repetitive TiO2 incubations of SCX-FT are not sufficient to detect all present multiphosphorylated peptides and that an additional, complementary dimension of fractionation is required for a comprehensive mapping of phosphopeptides in complex mixtures. Thus, we decided to systematically evaluate the combinatorial use of SCX and ERLIC. Combination of SCX−ERLIC Yields Higher Numbers of Multiphosphorylated Peptides Than SCX-Only

We could show in our previous work that, after fractionation by SCX, more than 93% of detected phosphopeptides were singly and doubly phosphorylated, which is likely due to charge− charge repulsion of multiply phosphorylated peptides from the SCX stationary phase.18 As we could show above, even multiple incubations with TiO2 beads do not lead to a comprehensive mapping of phosphopeptides present in SCX-FT. To overcome this limitation one can (1) fractionate the FT, or (2) reseparate the FT, for example, on an ERLIC column, to enrich multiphosphorylated peptides in different fractions. Fractionation of FT is a rather crude approach and, consequently, a second chromatography dimension which separates singly and 4272

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Figure 3. Distributions of identified phosphopeptides by SCX−ERLIC fractionation. (A) Percentages of all nonredundant identified mono- and multiphosphorylated peptides by SCX-ERLIC are depicted. (B and C) Numbers and percentages of nonredundant identified mono- and multiphosphorylated peptides by SCX (B) and after reseparation of SCX-FT by ERLIC (C) are shown. (D) Numbers of identified phosphopeptides and their distribution over the ERLIC gradient in the second dimension are shown. (E) Venn diagram of nonredundant phosphopeptides identified in both methods. Phosphopeptides are colored according to bound phosphate groups (1p to ≥4p).

3E). Median ion scores were similar for both approaches indicating as well that another class of peptides is enriched in the second dimension and not low-abundance “leftovers” picked up by chance (Table 1). This is also highlighted by peak intensities (Supporting Information Figures S2−S4). Peak intensities were highest for SCX used in the first dimension, which can be explained by the relatively high amount of singly phosphorylated peptides and its high binding capacity. ERLIC in the first dimension exhibited the lowest intensities, which is most likely due to the high number of multiphosphorylated peptides. Median peak intensities spread from 2.1 × 106 to 7.3 × 106 indicating good abundance of phosphopeptides in both chromatographic dimensions. We also compared the distribution and numbers of identified mono- and multiphosphorylated peptides by SCX−ERLIC to the described one-dimensional fractionation by SCX-only. In SCX-only, 429 multiphosphorylated peptides (≥3p) were identified, whereas by SCX−ERLIC, 845 multiphosphorylated peptides were identified, an increase by almost 49%. Moreover, by SCX−ERLIC, we identified 5300 nonredundant phosphopeptides, approximately 22% more than by SCX-only (Table 1). In conclusion, we demonstrated that the use of two complementary separation techniques accommodates in-depth phosphopeptide analysis and increases both the number of singly and multiply phosphorylated peptides identified from complex biological samples, in our case whole cell lysate, without elongating the MS analysis time.

doubly phosphorylated peptides in earlier fractions from multiphosphorylated peptides in later fractions seems to be advantageous. Thus, we fractionated peptides by SCX and reseparated the SCX-FT by ERLIC after adjustment of the organic solvent composition and pH (see Experimental Methods for details). Multiphosphorylated peptides bind strongly to the ERLIC column because of their higher number of negative charges and most of the singly phosphorylated peptides elute in early fractions. All fractions, the ones collected by SCX and the new ERLIC fractions, were incubated with TiO2 beads and eluted peptides were analyzed by RP-LC−MS/ MS. A total of 10 831 phosphopeptides were identified from 27 fractions (15 SCX and 12 ERLIC fractions), of which 5300 were nonredundant (Figure 3A; Table 1; Supporting Information Table S2). In 15 SCX fractions in the first dimension, 7013 phosphopeptides were detected of which 3454 were nonredundant. The majority of these, similar to our previous report, were singly phosphorylated (60.5%; Figure 3B). Analysis of the ERLIC fractions resulting from the reseparation of SCX-FT showed a remarkable difference in the number of detected multiphosphorylated peptides. More than 31% of detected nonredundant phosphopeptides carried more than two phosphate groups highlighting that a second complementary chromatographic dimension is crucial for comprehensive phosphopeptide analysis (Figure 3C). In agreement with our previous results, most singly phosphorylated peptides were present in the ERLIC-FT and the first fraction, whereas the majority of multiphosphorylated peptides were found in the last fractions (Figure 3D).18 The higher affinity of multiphosphorylated peptides to the ERLIC stationary phase is advantageous to avoid signal suppression of multiphosphorylated peptides by singly or nonphosphorylated peptides in MS analysis, hence, enhancing the detection of these species. In SCX−ERLIC, 59.7% of nonredundant identified phosphopeptides were detected only by SCX and 34.8% only by ERLIC. The low percentage of nonredundant phosphopeptides identified by both methods (5.5%) confirmed that these two approaches are highly complementary (Figure

ERLIC−SCX Fractionation Further Increases Numbers of Identified Phosphopeptides

To provide a more comprehensive view of different possible configurations of combinatorial fractionation approaches for phosphopeptide analysis, ERLIC−SCX was selected as an alternative to SCX−ERLIC. We used the same sample and instrumental setup as in the other approaches (Figure 1). Peptides were separated by ERLIC; the resulting ERLIC-FT was concentrated to reduce the amount of ACN to 30% and then subjected to a SCX column (see Experimental Methods for further details). All fractions were incubated by TiO2 beads and eluted peptides analyzed by RP-LC−MS. 4273

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Figure 4. Distributions of identified phosphopeptides by ERLIC−SCX fractionation. (A) Percentages of all nonredundant identified mono- and multiphosphorylated peptides by ERLIC-SCX are depicted. (B and C) Numbers and percentages of nonredundant identified mono- and multiphosphorylated peptides by ERLIC (B) and reseparation of ERLIC-FT by SCX (C) are shown. (D) Numbers of identified phosphopeptides and their distribution in each fraction over the SCX gradient in the second dimension are shown. (E) Venn diagram of nonredundant phosphopeptides identified in both methods. Phosphopeptides are colored according to bound phosphate groups (1p-4,5p).

Higher numbers of multiphosphorylated peptides in SCX− ERLIC could also be due to better fractionation of singly and doubly phosphorylated peptides by the SCX stationary phase (Supporting Information Figure S6). As especially singly phosphorylated peptide species are significantly reduced in subsequent ERLIC separations, the latter are more efficient. Multiphosphorylated peptides are fractionated in the absence of singly and doubly phosphorylated peptides further enhancing fractionation power and thus identification by MS analysis. We compared percentages of identified multiphosphorylated peptides in our combinatorial fractionation approaches with several methods that were recently developed to improve phosphoproteome analysis. Continuous pH elution of SCX-FT from a strong anion exchange (SAX) column yielded 58.9%, 31.4%, and 9.7% of singly, doubly, and triply phosphorylated peptides, respectively, which are almost identical to our results by SCX-only (Supporting Information Figure S1). Hence, continuous pH elution of phosphopeptides from SAX does not increase isolation and identification of multiphosphorylated peptides.21 In another study, an affinity-based strategy by successive elution of phosphopeptides from TiO2 microcolumns with various buffers was introduced to expand the phosphoproteome coverage.30 Among 1800 identified phosphopeptides, more than 89% were singly phosphorylated. In contrast, in both combinatorial approaches described here, ERLIC fractionations showed higher percentages of multiphosphorylated peptides (Figures 3C and 4B). In conclusion, based on our data, combinations of SCX and ERLIC yield complementary results and allow a comprehensive phosphoproteome analysis not discriminating between pSer-, pThr-, and pTyr-containing peptides (Table 1). Both approaches are highly orthogonal as indicated by the small overlap of identified phosphopeptides. This is also true for

A total of 13 486 phosphopeptides were identified from 27 fractions (15 ERLIC and 12 SCX fractions), of which 6433 were nonredundant (Figure 4A; Table 1; Supporting Information Table S5). In the first dimension, 6433 phosphopeptides were detected of which 3345 were nonredundant. A majority of detected phosphopetides were multiphosphorylated (≥85%; Figure 4B; Supporting Information Table S3), while a majority of phosphopeptides identified after SCX separation of the ERLIC-FT were singly phosphorylated (72%; Figure 4C,D). Thus, SCX−ERLIC yielded 15% more multiphosphorylated peptides (≥3p) than ERLIC−SCX (845 versus 718). However, with ERLIC−SCX, we identified 21% and 48% more nonredundant phosphopeptides compared to SCX−ERLIC and SCX-only, respectively, in the same LC− MS time (Table 1). Forty-eight percent of the nonredundant identified phosphopeptides were detected only by ERLIC and 46.4% only by SCX (Supporting Information Table S3). Similar to SCX−ERLIC, only 5.6% of identified phosphopeptides were shared between the two fractionation methods, again validating that the two methods are highly complementary (Figure 4E). Surprisingly, we identified more phosphopeptides by ERLIC when it was used in the first dimension than in the second dimension, or to ERLIC-only reported in our previous study.18 One possible explanation might be the use of a shorter gradient, which led to a broad distribution of doubly phosphorylated peptides over the entire ERLIC gradient. These peptides are easier to identify by CID-based MS/MS experiments than their higher phosphorylated counterparts. The presence of doubly phosphorylated peptides in later ERLIC fractions may also explain the lower identification rate of higher multiphosphorylated peptides in ERLIC−SCX compared to SCX−ERLIC as doubly phosphorylated peptides may suppress signals of higher phosphorylated species (Supporting Information Figure S5). 4274

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identified phosphosites, which overlapped by approximately 20% between the first and second chromatographic dimension (Supporting Information Table S4). ERLIC−SCX appears to be the better choice compared to SCX−ERLIC regarding the number of detected phosphopeptides and identified phosphosites (Figure 5). However, if the sample amount is high and

Article

AUTHOR INFORMATION

Corresponding Author

*(J.D.) Tel.: +49-761-203-97208. Fax: +49-761-203-8456. Email: [email protected]. (M.Z.) Tel.: +49-761203-97220. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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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, the Deutsche Forschungsgemeinschaft (GZ DE1757/2-1, DE1757/3-1), and from the Federal Ministry of Education and Research through GerontoSys II − NephAge (031 5896 A). We thank all Protein Dynamics group members, as well as M. Boerries, H. Busch and colleagues from the ZBSA, for helpful discussions and support.

Figure 5. Distributions of identified phosphopeptides in ERLIC−SCX, SCX−ERLIC, and SCX-only fractionation methods are shown.

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detection of multiphosphorylated peptides is essential, we recommend use of the SCX−ERLIC strategy which has several practical advantages. The loading capacity of a standard SCX column is higher than that of an ERLIC column, hence, requiring a smaller column for the same amount of sample. This in turn leads to a smaller FT volume which is favorable for sample loading in the second dimension. Moreover, SCX-FT can be diluted by ACN and directly applied onto an ERLIC column, whereas ERLIC-FT contains 70% ACN, which needs to be decreased to 30% to meet the requirements of SCX chromatography. This step is not only time-consuming, but may also result in sample loss.

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CONCLUSION In this study, we present the benefits of combining complementary fractionation methods for phosphopeptide analysis. We used SCX and ERLIC to enhance phosphoproteome coverage by utilizing advantages of both approaches. Both combinatorial methods SCX−ERLIC and ERLIC−SCX identified substantially higher numbers of phosphosites, phosphopeptides, and multiphosphorylated peptides than other reported MDC approaches without leading to longer MS acquisition times. The numbers of identified multiphosphorylated peptides (≥3p) from ERLIC−SCX and SCX−ERLIC were 40% and 50% higher, respectively, than by SCX-only. Interestingly, ERLIC−SCX and SCX−ERLIC yielded also higher numbers of singly and doubly phosphorylated peptides than SCX-only, again emphasizing the benefits of combinatorial LC approaches for large-scale phophoproteomics studies.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

Additional supporting figures S1−S6, 4 supporting tables with identified phosphopeptides (Supporting Table S1 [SCX-only], Supporting Table S2 [SCX−ERLIC], Supporting Table S3 [ERLIC−SCX]), and phosphosites (Supporting Table S4). This material is available free of charge via the Internet at http://pubs.acs.org. 4275

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Journal of Proteome Research

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