Selective Enrichment of Cysteine-Containing Phosphopeptides for

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Selective enrichment of cysteine-containing phosphopeptides for subphosphoproteome analysis Mingming Dong, Yangyang Bian, Jing Dong, Keyun Wang, Zheyi Liu, Hongqiang Qin, Mingliang Ye, and Hanfa Zou J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00830 • Publication Date (Web): 10 Nov 2015 Downloaded from http://pubs.acs.org on November 10, 2015

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Journal of Proteome Research

Selective enrichment of cysteine-containing phosphopeptides for subphosphoproteome analysis

Mingming Dong,a,b Yangyang Bian,a,b Jing Dong,a Keyun Wang,a,bZheyi Liu,a,b Hongqiang Qin,a Mingliang Ye,a,*Hanfa Zoua,*

a

Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic

Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; b *

University of Chinese Academy of Sciences, Beijing 100049, China To whom correspondence should be addressed, H.F. Zou: Phone: 86-411-84379610. Fax:

86-411-84379620. E-mail: [email protected]. M.L. Ye: Phone: 86-411-84379620. Fax: 86-411-84379620. E-mail: [email protected].

1

ACS Paragon Plus Environment


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ABSTRACT Among the natural amino acids, cysteine is unique since it can form disulfide bond through oxidation and reduction of sulfhydryl, and thus plays pervasive role in modulation of proteins activities and structures. Crosstalk between phosphorylation and other post-translational modifications has become a recurrent theme in cell signaling regulation. However, the crosstalk between the phosphorylation and the formation and reductive cleavage of disulfide bond has not been investigated so far. To facilitate the study of this crosstalk, it is important to explore the subset of phosphoproteome where phosphorylations are occurred near to cysteine in the protein sequences. In this study, we developed a straight forward sequential enrichment method by combining the thiol affinity chromatography with the immobilized titanium ion affinity chromatography to selectively enrich cysteine-containing phosphopeptides. The high specificity and high sensitivity of this method was demonstrated by analyzing the samples of Jurkat cells. This ‘divide and conquer’ strategy by specific analysis of a subphosphoproteome enables identification of more low abundant phosphosites than the conventional global phosphoproteome approach. Interestingly, amino acid residues surrounding the identified phosphosites were enriched with buried residues (L, V, A, C) while depleted with exposed residues (D, E, R, K). And the phosphosites identified by this approach showed a dramatically decrease in locating in disorder regions compared to that identified by conventional global phosphoproteome. Further analysis showed that more proline directed kinases and less acidophilic kinases were responsible for the phosphorylation sites of this subphosphoproteome. Key words: Subphosphoproteome analysis, cysteine-containing phosphopeptide, crosstalk

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INTRODUCTION The biological functions and structures of almost all eukaryotic proteins are fine-tuned by various post-translational modifications (PTMs).1, 2 The interplay and co-regulation of different PTMs have drawn much more attention than before.3,

4

Among the 20 natural amino acids, cysteine is quite unique since it is the active sites of redox and can form disulfide bond through oxidation and reduction of sulfhydryl, thus it plays a pervasive role in modulation of protein activities and structures.5 Reversible protein phosphorylation, on the other hand, is among the most important and best explored PTMs, plays a pivotal role in almost all biological processes including cell division, differentiation, polarization and apoptosis.2 Moreover, it functions as molecular switches in cellular signal transduction. Crosstalk between phosphorylation and other post-translational modifications has become a recurrent theme in cell signaling regulation. For example, Swaney et al. performed the large scale analysis of the cross-talk between phosphorylation and ubiquitylation during protein degradation,6 and demonstrated that distinct phosphorylation sites were often used in conjunction with ubiquitylation and that these sites were highly conserved. However, the crosstalk between phosphorylation and the formation and reductive cleavage of disulfide bond has not been investigated so far. As the active sites of redox, cysteine often plays critical roles in key functional protein such as protein kinase and phosphatase. To facilitate the study of the crosstalk between phosphorylation and disulfide bond, and also to characterize the phosphorylation events of the key functional cysteine-containing proteins, it is important to explore the subset of phosphoproteome where phosphorylations are occurred near by cysteine in the protein sequences. Recent advances in mass spectrometry (MS)-based proteomics have made it possible to identify ten thousands of phosphosites from a cell or tissue sample per study.7, 8 However, identification of low abundant phosphosites is still a big challenge due to the low abundance of phosphoproteins and the low stoichiometry of the 3



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phosphorylation. The problem with shot-gun proteomics is that the peptides produced in the proteolysis step overwhelm the analytical capacity of current LC/MS systems, both in number and in dynamic range. Accepting the fact that complete proteome analysis will remain difficult using currently available methods, a ‘divide and conquer’ strategy has emerged to comprehensively analyze specific subsets of the proteome that are selectively isolated.9 Similarly, the ‘divide and conquer’ strategy could also be applied in phosphoproteome analysis to identify low abundant phosphosites. The subphosphoproteome approach by selective enrichment of pTry (phosphorylated tyrosine) peptides was proved to identify more pTry sites than the global phosphoproteome approach where all phosphopeptides were enriched.7, 10, 11 A negative enrichment strategy by depletion of a subset of phosphopeptides, i.e. acidic phosphopeptides was reported to enhance the identification of basophilic kinase substrates.12

Selective

enrichment

of

mono-phosphopeptides

or

multi-phosphopeptides was proven to improve the coverage for that specific type of subphosphoproteome.13 Therefore, as a subphosphoproteome approach, selective enrichment of the phosphopeptides containing cysteine residue also has the potential to identify low abundant phosphosites. We term this subphosphoproteome as Cys-subphosphoproteome since these phosphosites neighboring with cysteine. For analyzing the Cys-subphosphoproteome, the key is to selectively enrich cysteine-containing phosphopeptides. In this study, we developed a straight forward method to do this. Firstly, proteins were reduced by Dithiothreitol (DTT) and digested by trypsin. Then the acquired peptides were incubated with thiol-specific affinity resins, and the cysteine-containing peptide could be enriched with high specificity. The cysteine-containing peptides were further incubated with immobilized titanium (IV) ion affinity chromatography (Ti4+-IMAC) beads to enrich phosphopeptides. This subphosphoproteome approach identified three times more cysteine-containing phosphopeptides than that achieved by the conventional global phosphoproteome approach. It was found that amino acid residues surrounding the phosphosites identified by Cys-subphosphoproteome approach were enriched with buried residues while depleted with exposed residues. Based on the kinase substrates predicted by 4


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bioinformatics tool, we found more proline directed kinases and less acidophilic kinases were responsible for the phosphorylation of residues neighboring cysteine. MATERIALS AND METHODS Materials and chemical reagents All water used in this experiment was prepared using a Milli-Q system (Millipore, Bedford, MA). Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (NH4HCO3), trifluoroacetic acid (TFA), sodium orthovanadate (Na3VO4), sodium fluoride (NaF), trypsin were all obtained from Sigma Aldrich (St. Louis, MO, USA). Formic acid (FA) was bought from Fluka (Buches, Germany), and acetonitrile (ACN, HPLC grade) was purchased from Merck (Darmstadt, Germany).

Fused-silica

capillaries with 200 µm i.d. and 75 µm i.d. were purchased from Polymicro Technologies (Phoenix, AZ, USA). Daisogel ODS-AQ (5 µm, 12 nm pore) was purchased from DAISO Chemical CO., Ltd. (Osaka, Japan). The Thiopropyl Sepharose® 6B was purchased from GE healthcare (Shanghai, China). Cell Culture and Digest Preparation The Jurkat cells were grown in RPMI-1640 (Roswell Park Memorial Institute 1640), supplemented with 10% bovine serum, 100 U/mL of streptomycin and penicillin. The cells were harvested at about 80% density. Half amount of the cells were treated with 1mM freshly prepared pervanadate for 15 min at 37 ℃, and the other ones were left untreated. After that, the cell pellets were softly homogenized in an ice-cold lysis buffer containing 8 M urea, 50 mM Tris-HCl (pH = 7.4), 2% protease inhibitor cocktail (v/v), 1% Triton X-100 (v/v), 1 mM NaF, and 1 mM Na3VO4, sonicated at 400 W for 120 s, and centrifuged at 25, 000 g for 1 h. The proteins were precipitated and purified with ice-cold acetone/ethanol/acetic acid, as described in our previous study14. Then, the proteins were dissolved in a buffer containing 100 mM NH4HCO3 (pH = 8.2) and 8 M urea. After reduced by DTT at 37 ℃ for 2 h, the samples were diluted to 1 M urea and digested by trypsin with an enzyme-to-protein ratio of 1/25 (w/w). The resulted peptide mixture was desalted by OASIS HLB column (Waters, Milford, MA, USA). 5



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Cysteine-containing peptide enrichment and Phosphopeptide Enrichment The enrichment of cysteinyl containing peptide was performed as described by Liu15 et al. Briefly, tryptic digest from 1 mg protein was dissolved in 100 µL 50 mM Tris buffer, pH 7.5, 1 mM EDTA (coupling buffer) and reduced by 5 mM DTT at 37 ℃ for 30min. Then the sample was diluted to 500 µL by adding coupling buffer and was incubated with 100 µL of Thiopropyl Sepharose 6B thiol-affinity resin for 2h at 25℃. After that the resin was washed in turn with 0.5 mL×5 of 50 mM Tris buffer, pH 8.0 (washing buffer), 1M NaCl, 80% ACN，and coupling buffer. The resin was incubated triple with 100 µL of 20 mM freshly prepared DTT to release the captured cysteinyl-peptides. Then the sample was alkylated with 80 mM iodoacetamide for 30 minutes at RT in the dark. Part of the sample was cleaned by C18 SPE column and analyzed by LC-MS/MS. The other was further enriched with immobilized titanium (IV) ion affinity chromatography (Ti4+-IMAC) for phosphopeptide enrichment. The enrichment of phosphopeptides was performed as described previously.16 Briefly, peptide mixtures were first incubated with Ti-IMAC beads at a ratio of 1:10 w/w in loading buffer (80% ACN and 6% TFA). After centrifugation, the supernatant was removed. The Ti-IMAC beads with adsorbed phosphopeptides were then washed in turn by two washing buffers (50% ACN, 6% TFA containing 200 mM NaCl as washing buffer 1, 30% ACN containing 0.1% TFA as washing buffer 2) to remove nonspecific adsorbed peptides. The bound phosphopeptides were then eluted by 10% NH3·H2O. After centrifugation at 20000 g for 5 min, the supernatant was collected and lyophilized to dryness. Nano LC-MS/MS analysis The nano RPLC−MS/MS-experiments were performed on an UltiMate 3000 RSLCnano systems (Thermo Scientific, USA) connected to a Q Exactive (Thermo Scientific, USA). The samples were dissolved in 0.1% FA, 6 µL of each sample was automatically loaded onto the C18 trap column (3 cm × 200 µm i.d.) at a flow rate of 5 µL / min. The 75 µm i.d. analytical column was packed with C18 AQ particles (5 µm, 12 nm) to 15 cm length. The mobile phase A was 99.9% water / 0.1% FA, and mobile phase B was 80% ACN / 0.1% FA. The elution gradient executed was 5% to 6


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35% mobile phase B lasted for 78min. The Q Exactive instrument was operated in the data dependent mode. Survey scan MS spectra (m/z 400−2000) were acquired by the Orbitrap with 70 000 resolution (m/z 200), and the AGC target was set to 1 × 106 with a max injection time of 120 ms. Dynamic exclusion was set to 30 s. The 12 most intense multiply charged ions (z ≥ 2) were fragmented by higher-energy collisional dissociation (HCD). The MS/MS scans were also acquired by the Orbitrap with 35 000 resolution (m/z 200), and the AGC target was set to 1× 105 with a max injection time of 120 ms. Typical mass spectrometric settings were as follows: spray voltage, 2 kV; heated capillary temperature, 250 °C; normalized HCD collision energy 27%. Database Search and Data Analysis The raw data files generated by the Q Exactive were searched with software MaxQuant version 1.3.0.5,17 against the uniprot human database (released on December 11, 2013 and containing 88473 protein sequences), supplemented by frequently observed contaminants, and reversed versions of all sequences were contained. Enzyme specificity was set to trypsin (KR/P), up to two missed cleavage sites were allowed. Phospho (STY), oxidation (M), loss of ammonia and water were chosen for variable modifications, carbamidomethyl was set as fixed modifications. The maximum false-discovery rate (FDR) was set to 1% for both the peptides and proteins. The minimum required peptide length was set at six amino acids. A web-based application Two Sample Logo18 was used to calculate and visualize differences between two sets of aligned samples of amino acids. Motif-X algorithm19 (http://motif-x.med.harvard.edu) was also used to generate phosphorylation motifs for the identified phosphorylation sites. The web server IUPred20 was used to predict the disorder tendency for the identified phosphorylation sites, with the prediction type set as short disorder. RESULTS AND DISCUSSION As the main goal of this study was to analyze the Cys-subphosphoproteome, where phosphosites neighboring with cysteine residue, it’s of interest to know how 7



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many cysteine-containing phosphopeptides present in the dataset acquired by conventional phosphoproteomics approach. Therefore, we performed a global phosphoproteome analysis of Jurkat cells by conventional phosphopeptide enrichment approach where a single-step Ti4+-IMAC enrichment was performed. The three replicate LC MS/MS analysis of the enriched phosphopeptides resulted in the identification of 7346 phosphopeptides. It was found that only 573 of the 7346 phosphopeptides (less than 8%) contained cysteine (details were provided in Table S-1 and Table S-3), indicating the low abundance of cysteine-containing phosphopeptides.

Thus

phosphopeptides

is

the

selective

indispensable

for

enrichment

of

comprehensive

cysteine-containing analysis

of

the

Cys-subphosphoproteome. Theoretically this could be achieved by combining the thiol affinity chromatography with the Ti4+-IMAC enrichment, and these two enrichment steps should be proceeded in sequence. Phosphopeptides captured on the Ti4+-IMAC resins could be eluted by volatile ammonium solution and then lyophilized for direct RPLC MS/MS analysis, thus had good compatibility with MS analysis. Considering this, we adopted the workflow shown in Figure 1 where the thiol affinity chromatography was performed before Ti4+-IMAC for the selective enrichment of cysteine containing phosphopeptides. This

sequential

enrichment

method

was

applied

to

analyze

the

Cys-subphosphoproteome of Jurkat cells. We first investigate the performance of the thiol-affinity chromatography for the enrichment of cysteine-containing peptides. Proteins derived from pervanadate treated Jurkat cells were reduced with DTT and digested by trypsin, as shown in Figure 1. The resulting peptides were reduced by 5 mM DTT again to break down the scrambling disulfide bonds that may formed by the free sulfydryl groups on cysteine residues at alkaline pH during protein digestion. Then the peptide mixtures were incubated with a commercially available thiol-affinity resin (thiopropyl sepharose 6B) for the enrichment of cysteine-containing peptides. The eluted peptides were subjected to LC MS/MS analysis by Q-Exactive in triplet. The three raw data files were combined together and searched by MaxQuant. After controlling the false-discovery rate (FDR) < 1%, we identified 2435 peptides in total, 8


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of which 99% (2412) contained at least 1 cysteine in the peptide sequence. For comparison, the same tryptic digest sample without thiol-affinity enrichment was directly analyzed by LC MS/MS in triplet. Of the 2202 peptides identified, only 19 % of them containing cysteine. Details of the identified peptides were provided in Table S-4. Above data indicated the high specificity of thiol-affinity enrichment. The eluted peptide mixture from the first step thiol-affinity enrichment was then subjected to Ti4+-IMAC enrichment (Figure 1). The acquired phosphopeptides by this sequential enrichment method were analyzed in triplet by LC MS/MS, and the corresponding raw files were combined for database searching by MaxQuant. In total, we identified 2089 unique phosphopeptides, among them 1919 (92%) had at least one cysteine in their sequence. Compared to the global phosphoproteome, the percentage of cysteine-containing phosphopeptide was increased by around 12 folds (Figure 2.A and B). The high quality result we obtained benefit from the high specificity of both the thiol-affinity enrichment and the phosphopeptide enrichment. Next,

we

compared

the

phosphopeptides

identified

by

the

global

phosphoproteome approach (single step Ti-IMAC enrichment) with that identified by the Cys-subphosphoproteome approach (sequential enrichment). It was found that 352 phosphopeptides overlapped in both datasets, account for only 4% of the total phosphopeptides.

And

1734

phosphopeptides

(of

which

1615

were

cysteine-containing phosphopeptides) were newly identified by the sequential enrichment method, which illustrating the ‘divide and conquer’ strategy allowed the identification of phosphopeptides with lower abundance. Though the Cys subphosphoproteome approach identified much less phosphopeptides than the global phosphoproteome approach did (7346 vs. 2089), it identify three times more cysteine-containing phosphopeptides (573 vs. 1919), and two third of the cysteine-containing phosphopeptides identified by the globe phosphoproteome approach can also be identified by the Cys-subphosphoproteome approach as shown in Figure 2.C. Clearly this sequential enrichment approach significantly improved the coverage for Cys-subphosphoproteome analysis. This approach was applied to analyze the Cys-subphosphoproteomes of two other 9



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Jurkat cell samples. One (Sample 2) was treated with pervanadate as the sample analyzed above (Sample 1), and the other one (Sample 3) was not treated with pervanadate. The enriched phosphopeptides were analyzed in triplet by LC MS/MS as above, which leaded to the identification of 2106 and 2046 unique cysteine-containing phosphopeptides, respectively, with the specificity higher than 80% (Table S1). Above

data

confirmed

that

this

approach

could

consistently

identify

cysteine-containing phosphopeptides. In terms of phosphosites, 1658, 2165 and 1823 sites were identified from Sample 1, 2 and 3, with the percentage of pTyr (phosphorylated tyrosine) sites accounted for 9%, 5% and 1%, respectively. Detail information was provided in Table S-2 and S-5. Higher percentages of pTyr sites were identified from the first two samples because sodium pervanadate was the inhibitors of tyrosine phosphatase. From these three samples we totally identified 3195 phosphosites, among them 1826 were high confident sites (with localization probability > 0.75 and score difference > 5). Among the high confident sites ,1463 sites were found to be with at least 1 cysteine residue locating within ±15 amino acid residues surrounding the phosphosite (set as 0). This high confident dataset was then used to characterize the Cys-subphosphoproteome. To get a straightforward visualization of the difference between phosphosites identified by the two approaches, i.e. the Cys-subphosphoproteome approach and the global phosphoproteome approach, the on-line software, Two Sample Logo18, was applied to analyze the aligned sequences of the identified phosphosites. The 1463 phosphosites neighboring with Cysteine identified by the Cys-subphosphoproteome approach were set as positive dataset, and phosphosites identified by the global phosphoproteome approach were set as negative dataset. From the results shown in Figure 3, we can see that the distribution of Cysteine around the phosphosites was not even. For pSer/pThr sites, Cysteine was enriched on the position of -2 and -1. The pTyr sites showed a quite different pattern, where Cysteine was largely enriched on the +1 position. Cysteine is typically considered as one type of the buried residues in globular proteins.21 It’s not surprise that Cysteine (C) is enriched on sites surrounding the phosphosites in the Cys-phosphoproteome since the cysteine containing 10


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phosphopeptides were enriched for identification. Interestingly, other buried residues such as L,V, A were enriched in the Cys-subphosphoproteome while the exposed residues such as D, E, R, K were depleted. This is especially true for pSer sites. This hinted that there may be subtle difference in the preference of the kinases to recognize these phosphosites. The Motif-X (http://motif-x.med.harvard.edu) is a software tool designed to extract overrepresent patterns from any sequence data set. In this study, it was used to extract the overrepresent motifs from the Cys-phosphoproteome and the global phosphoproteome using the human fasta database as background (Figure S-1 and Figure S-2), respectively. As we know, the Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. After comparing Figure S-1 and Figure S-2, we found that proline-directed kinase substrates (with a proline shown on the +1 position） were largely enriched by the sequential cysteine-containing phosphopeptide enrichment method, with a cysteine simultaneously shown in the motif. Further analysis showed that the top 9 motifs generated by such phosphosites were pSP with a cysteine shown within the -7 to +7 position, as shown in Figure S-1. There were two motifs with the presence of basophilic residue Arginine (R) and cysteine, RXXpSXXC and RCXpS. Phosphopeptides originating from acidic kinase substrates always made up a large proportion in the conventional phosphopeptide enrichment method, and many motifs with D/E were observed for global phosphoproteome (Figure S-2). However, after performing the cysteine-containing phosphopeptide enrichment, it was hard to find a motif with both acidic residues D/E and C in a single motif. This is consistent with the fact that acidic residues D/E are depleted in the Cys-subphosphoproteome as discussed above. Identification of phosphorylation sites with their cognate protein kinases is of great importance in understanding signal transduction in complex biological systems. To obtain the putative kinase information of the phosphorylation sites identified, GPS22 (Group-based Prediction System) was used to predict kinase-specific phosphorylation sites at a high stringency level. The bar chart in Figure 4.A showed 11



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the relative distribution of predicted kinase-specific substrates found in global phosphoproteome (blue bar) and Cys-subphosphoproteome (red bar). From the result, we can see that the CMGC kinase group substrates identified in the Cys-subphosphoproteome increased notably, from 27% to 38%, as compared to that identified in the global phosphoproteome. On the contrary, phosphopeptides originating from acidic kinase substrates like Casein kinase 1 (CK1), and basophilic AGC kinase group (including protein kinase A, protein kinase G, and protein kinase C et al.) showed a decrease in the Cys-subphosphoproteome. The CMGC kinase group included a series of key kinases like Mitogen-activated protein kinases (MAPK), cell cycle cyclin dependent kinases (CDK), and kinases involved in splicing and metabolic control.

The

enrichment

of

CMGC

kinase

group

substrates

in

the

Cys-subphosphoproteome indicated the crosstalk between phosphorylation and disulfide bond formation and dissociation may play an important role in regulating biological processes. Previously it was found that phosphorylated Ser, Thr, and Tyr residues exhibited notably differences in structural classification compared to their unmodified counterparts, they were predominantly predicted to locate in intrinsically disordered regions rather than in ordered secondary structures.23 Recent research show that tyrosine phosphorylation is kind of different from phosphorylated Ser/Thr, is often observed in ordered interface regions which are not predicted to be disordered in the unbound state.24, 25 Intrinsically disordered regions are flexible and extended protein segments that have no ordered secondary structure under physiological conditions, they featured their sequence by rich in disorder-promoting residues (D, K, R, S, Q, P, and E) and devoid of order-promoting residues (W, Y, F, I, V, L, and T). Despite a large number of phosphorylation sites show a preference to locating in disordered regions, there are also phosphosites undertake important functions located in the structured regions. For example, Gygi et al found in their work that most of the 120 observed activation loop phosphosites of kinase were ordered, with elevated levels classified as strands.26 For our result, the buried residues (L, V, A, C) were enriched while the exposed 12


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residues (D, E, R, K) were depleted in the Cys-subphosphoproteome also implies these phosphosites may have some connection with protein structure. We used a web-based software IUPred to predict the disorder/order tendency for the identified phosphosites, and the results were shown in Figure 4.B. It was found that 85.8% of pSer/pThr sites in global phosphoproteome were predicted to locate in disorder regions, while only 46.1% phosphorylated Tyr sites were located in disorder regions. This was in consistent with the knowledge that pSer/pThr sites were more frequently observed in disordered regions compared with pTyr sites. After performing the cysteine-containing phosphopeptides enrichment, the identified phosphorylated Ser/Thr/Tyr sites all showed a dramatically decrease in locating in disorder regions. The pSer/pThr sites decreased from 85.8% to 59.5%, and the pTyr sites decreased from 46.1% to 18%. Collectively, the order-promoting cysteine affects the micro-environment of the phosphorylation site, and a subclass of phosphorylation sites which tend to located in ordered regions of the protein were identified. Above analysis indicated that there are some differences between global phosphoproteome and Cys-subphosphoproteome. However, we do not know if there is a biological significance for the phosphorylation occurred close to Cysteine residues yet. It is well known that both Cys and phosphorylation play critical roles in key functional proteins such as protein kinase and phosphatase. A bioinformatic analysis of the human kinome27 revealed that there are 46 kinases that have a conserved Cys residue located in the ATP-binding site, and there are approximately 200 different kinases that have a cysteine located nearby the ATP pocket, suggesting cysteine may play important roles in regulation of enzyme activities. Indeed, an important class of kinase inhibitors is capable of forming an irreversible, covalent bond to the kinase active site by reacting with a cysteine residue,28 thus blocks the binding of ATP to the kinase, thereby rendering the kinase inactive. The activities of kinases are also regulated by the phosphorylation of the activation loop.29 Take the 90-kDa ribosomal S6 kinase 1 (Q15418) for example, the S221 phosphorylation site was identified in our dataset with an adjacent C223. Those two residues are located in N-terminal kinase domain. It was reported that this kinase can be activated by 13



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phosphorylation at S221 by PDK1 (3-phosphoinositide-dependent protein kinase 1).30, 31

It was also reported that the C223 is the active-site cysteine residue, and the

S-glutathionylation of C223 is both necessary and sufficient for the inhibition of this kinase during oxidative stress.32 Clearly the phosphorylation of S221 and oxidative state of C223 both have close relationships with the activity of this kinase. Protein-tyrosine phosphatases (PTPs) are another kind of important functional proteins that can dephosphorylate phosphotyrosyl residues of proteins and peptides. PTPs are also phosphoproteins. Intradomain phosphorylation modulates the catalytic activity of the PTP domain.33,

34

On the other hand, the hallmark of PTPs is an

essential cysteine residue at the catalytic site, which forms a thiol-phosphate intermediate during catalysis. The oxidation of the catalytic Cys residue causes the inactivation of the enzyme.35 In our dataset, we identified the Y50 sites with an adjacent C46 in the protein tyrosine phosphatase type IVA 2 (Q12974).36 The active site of this PTP was C101, which was confirmed to form disulfide bond with C46. The formation of disulfide bond may lead to the inactivity of the phosphatase. Considering the important roles of Cys and phosphorylation in regulating enzyme activities, we believe there is some type of crosstalk between these two modifications. The identification of the peptides that contain both Cys and phosphorylated groups will facilitate the study of this crosstalk. CONCLUTION In this study, we developed a sequential enrichment method to selectively enrich cysteine-containing phosphopeptides. The high specificity and high sensitivity of this method was demonstrated by analyzing the samples of Jurkat cells. This “divide and conquer” strategy by specific analysis of Cys-subphosphoproteome enables the identification of low abundant phosphosites neighboring cysteine residues. The obtained datasets allow the characterization of this subphosphoproteome for the first time. Interestingly, amino acid residues surrounding the phosphosites in this subphosphoproteome were enriched with buried residues while depleted with exposed residues. It was also found that more proline directed kinases and less acidophilic 14


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kinases are responsible for the phosphorylation sites. This is the first study to explore the relationship between the cysteine and phosphorylated S/T/Y, and will shed some light on the crosstalk between the form of disulfide bond and phosphorylation, also supply a tool for characterizing the phosphorylation events of key functional cysteine-containing proteins. We believe that the true relationship between disulfide bond and phosphorylation is quite complex, and its study depends not only the combination of bottom-up proteomics and top-down proteomics, but also the application of bioinformatics tools. And our method could be one of the important tools in the toolbox to decipher this important crosstalk. ACKNOWLEDGMENTS This work was supported by the China State Key Basic Research Program Grant (2013CB911204, 2012CB910101, and 2012CB910604), the Creative Research Group Project of NSFC (21321064), and the National Natural Science Foundation of China (21235006, 21275142, 81361128015, 21535008, and 21525524).

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FIGURE CAPTIONS Figure 1. Workflow for the analysis of Cys-subphosphoproteome by a sequential enrichment method. Figure 2. Cys-subphosphoproteome approach identified more Cys-containing phosphopeptides. The pie graphs (A) and (B) for phosphopeptides identified by the global and the Cys-subphosphoproteome approaches; (C) Overlap of Cys-containing phosphopeptides identified by the two methods. Figure 3. Two Sample Logos showed the differences between phosphosites identified by the Cys-subphosphoproteome approach and the global phosphoproteome approach. The aligned sequences centered with phosphosites were used to generate Logos, with phosphosites

identified

in

the

Cys-subphosphoproteome

and

the

global

phosphoproteome set as positive and negative dataset, respectively. Figure 4.

The differences of the phosphosites in Cys-subphosphoproteome and

Global phosphoproteome. (A) Bar chart shows the relative distribution of predicted kinase-specific substrates found in global phosphoproteome (blue bar) and Cys-subphosphoproteome (red bar). (B) Ratio of phosphosites identified in the global phosphoproteome (blue bar) and Cys-subphosphoproteome (red bar) locating in disorder regions.

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Overview of Supporting Information (Table S-1, Table S-2, Figure S-1, Figure S-2 are given in a Word file, Table S-3, Table S-4, Table S-5 are given in separate Excel files). Table S-1. Number of peptides identified by the Cys-subphosphoproteome approach or the Global phosphoproteome approach. Table S-2. Number of peptides identified by three independent sequential enrichment experiments. Table S-3. Phosphopeptide identified by the single-step Ti4+-IMAC. Table S-4. Peptides identified from tryptic digests of Jurkat cells or after performing thiol affinity chromatography enrichment. Table S-5. Phosphopeptides identified by three independent sequential enrichment experiments. Figure S-1. Motifs generated by the Cys-subphosphoproteome. Figure S-2. Motifs generated by the Global phosphoproteome.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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For Table of Contents Only

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Selective Enrichment of Cysteine-Containing Phosphopeptides for

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