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Molecular & Cellular Proteomics 3:279-286, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Dataset
Identification of Phosphoproteins and Their Phosphorylation Sites in the WEHI-231 B Lymphoma Cell Line*
From the Protein Chemistry Laboratory, Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX 75390-9196
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.
Knowledge about covalent modifications and their regulation is essential for the understanding of protein function. Regulation of protein activity is often modulated by reversible phosphorylation, and information about specific sites of phosphorylation is vital for understanding cellular signaling pathways. Two-dimensional gel electrophoresis is still the most common method used for detecting large-scale changes in phosphorylation (1). However, this method is time consuming and has a number of significant limitations. Although mass spectrometry is a sensitive tool for the identification of phosphopeptides, their detection within a complex peptide mixture can be limited by weak ionization of phosphopeptides (2). Due to the low stoichiometry of phosphorylation and the low abundance of signaling proteins within cells, enrichment of the phosphoproteins or phosphopeptides is often necessary. The ability to isolate phosphopeptides by immobilized metal affinity chromatography (IMAC)1 was first recognized by Andersson and Porath in 1986 (3). Recently, the use of IMAC in combination with mass spectrometry has allowed the identification of hundreds of phosphorylation sites in yeast (4) and Arabidopsis plasma membrane proteins (5).
An important goal of the Alliance for Cellular Signaling (AfCS) is the global analysis of ligand-induced changes in protein phosphorylation (6). Important steps in this process are the identification of phosphoproteins present in the AfCS model cell systems and the determination of their sites of phosphorylation. This information will be used to generate probes to obtain quantitative information on the effects of ligands on phosphorylation of specific sites. Based on recent progress in the application of IMAC and mass spectrometry, we used this approach to identify phosphoproteins and their phosphorylation sites in the murine WEHI-231 B lymphoma cell line.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Protein Test Samples—
One test sample (prepared at a ratio of 1:1:5) contained 1 pmol of a synthetic phosphotyrosine (p-Tyr) phosphopeptide (m/z 1127, DRVpYIHPF), a tryptic digest of 1 pmol of
-casein (containing three phosphopeptides: m/z 1467, TVDMEpSTEVFTK; m/z 1662, VPQLEIVPNpSAERR; and m/z 1953, YKVPQLEIVPNpSAERR), and a tryptic digest of 5 pmol each of four nonphosphorylated proteins (bovine serum albumin, carbonic anhydrase, ubiquitin, and ß-lactoglobulin). A second test sample was prepared with the same tyrosine phosphopeptide, -casein, and nonphosphorylated proteins as in the first test sample, but at a ratio of 1:2:400.
Cells—
Murine WEHI-231 B lymphoma cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 1 mM sodium pyruvate, and 20 mM HEPES. The cells were either left untreated or treated with 100 nM calyculin A (CLA) for 45 min. For experiments in which phosphopeptides were isolated by IMAC, 1 x 108 cells were lysed and the protein was isolated using 10 ml of TriPure reagent according to the manufacturer’s protocol (Roche Applied Science, Indianapolis, IN). The protein pellet was resuspended in 6 M guanidine hydrochloride at a ratio of 100 µl per mg of protein pellet. Alternatively, WEHI-231 cells (2 x 108) were collected by centrifugation and lysed in 1 ml per 4 x 107 cells of SDS lysis buffer (0.1% SDS, 150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 1 mM sodium orthovanadate). After boiling for 5 min, the SDS lysates were centrifuged at 100,000 x g for 1 h at 4 °C.
IMAC Column Preparation—
IMAC microtip columns were prepared as previously described (7). Twenty microliters of metal-chelating resin (50% slurry) was pipetted into the microtip columns. The microtip columns were then charged by applying 200 µl of 100 mM GaCl3 or FeCl3. The metal-chelating resins tested included POROS 20 MC (catalog no. 1-5428-02; Applied Biosystems, Foster City, CA); nitrilotriacetic acid-superflow (catalog no. 30510; Qiagen, Valencia, CA), and BRX (UNOsphere)-IDA (currently in beta-testing; catalog no. 3456-40; Bio-Rad, Hercules, CA).
Enrichment and Identification of Proteins by IMAC and Liquid Chromatography (LC)-Tandem Mass Spectrometry (MS/MS)—
Proteins in either 6 M guanidine hydrochloride or SDS lysis buffer were diluted 10-fold in 100 mM ammonium bicarbonate prior to digestion with 20 µg of trypsin per mg of protein overnight at 37° C. The peptides were desalted using a C18 cartridge, and 
1 mg of protein was loaded onto the activated IMAC columns. The IMAC columns were washed first with 0.1% acetic acid, then with 50% acetonitrile/0.1% acetic acid, then with 50% acetonitrile/0.1% acetic acid/100 mM sodium chloride, and finally with 0.1% acetic acid. The phosphopeptides were eluted with 20 µl of 200 mM Na2HPO4. Proteins were identified by LC-MS/MS using a nanoscale C18 column coupled in-line with an ion trap mass spectrometer (LCQ DECA; Thermo Finnigan, Inc., Woburn, MA). The instrument was run in data-dependent mode, cycling between one full MS scan and MS/MS scans of the four most abundant ions. The MS and MS/MS data were used to search the nonredundant NCBI mouse protein database using SEQUEST software. Software parameters were set to detect a modification of 80 Da on Ser, Thr, or Tyr. The assignments of phosphopeptide sequences were then manually confirmed by comparing the acquired MS/MS spectra to the theoretical fragmentation patterns.
Phosphothreonine (p-Thr) Immunoblotting—
Lysates from control and calyculin A-treated WEHI cells were immunoblotted with an anti-phosphothreonine antibody. WEHI-231 cells were either left untreated or treated with 20 nM CLA for 45 min and lysed in SDS sample buffer. The proteins (20 µg) were resolved on a 10% SDS gel, transferred to a nitrocellulose membrane, and immunoblotted with anti-phosphothreonine antibody (p-Thr-polyclonal) according to the manufacturer’s protocol (catalog no. 9381; Cell Signaling Technology, Beverly, MA).
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In order to optimize the identification of phosphopeptides, different metal-chelating resins and metal ions were tested for their ability to enrich phosphopeptides from a test sample composed of a 1:1:5 ratio of a p-Tyr phosphopeptide, phosphorylated
-casein peptides, and peptides derived from four nonphosphorylated proteins. POROS 20 MC and BRX-IDA resins charged with Ga3+ resulted in the lowest background of nonphosphorylated peptides and the highest recovery of each of the four phosphopeptides (data not shown). In general, resins charged with Ga3+ resulted in a higher signal-to-noise ratio with the standard mixture than the resins charged with Fe3+. In order to test the IMAC procedure under more rigorous conditions, a test sample containing the same mixture of peptides at a ratio of 1:2:400 was used. The test sample was loaded onto a Ga3+-charged BRX-IDA column, and samples from each step of the procedure were monitored using matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (Fig. 1). The phosphopeptides could not be detected in the total digest due to the abundance of nonphosphorylated peptides. The phosphopeptides were also not detected in either of the wash fractions, indicating they remained bound to the column. Analysis of the material eluted from the IMAC column with 200 mM Na2HPO4 showed that all four phosphopeptides were recovered with very little contamination by nonphosphorylated peptides.
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To maximize detection and identification of cellular phosphoproteins, proliferating WEHI-231 cells were treated with the serine/threonine phosphatase inhibitor CLA (8, 9). CLA is a member of a family of natural toxins that selectively inhibit the protein phosphatase 1 and 2A classes of serine/threonine phosphatase (10). Immunoblotting cell lysates with an anti-p-Thr antibody showed that CLA induced a robust increase in threonine phosphorylation in WEHI-231 cells (Fig. 2). For experiments in which phosphopeptides were isolated by IMAC, WEHI-231 cells were either left untreated or treated with 100 nM CLA for 45 min and lysed in either TriPure reagent or SDS Lysis buffer. The proteins isolated using TriPure were solubilized in 6 M guanidine-HCl. After trypsin digestion, the peptides were desalted prior to enrichment by IMAC. The peptides were eluted from the IMAC resin, loaded onto a capillary HPLC C18 column, washed to desalt, and coupled on line with an ion trap mass spectrometer. The peptides were resolved by gradient elution and identified by tandem mass spectrometry. The proteins identified in five experiments are listed in Table I. A total of 107 proteins were identified. Forty-two of the identified proteins were previously characterized phosphoproteins (PubMed links to relevant publications are included in the table). Fifty-four were known proteins not previously reported to be phosphorylated, and 11 of the proteins were "novel proteins" that have only been predicted from genomic or cDNA sequences.
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One hundred ninety-three distinct phosphorylation sites were identified. Only one of the sites identified was a phosphorylated tyrosine residue (dipeptidylpeptidase 8). All the others were serine or threonine phosphorylation sites. Of the 147 distinct phosphopeptides identified, 62 contained more than one phosphorylated amino acid and 85 had a single phosphorylated residue. Based on the relative numbers of acidic (Asp, Glu) and basic (Lys, Arg) residues present in the phosphopeptides, 75 were acidic, 49 were basic, and 23 were neutral.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The major goal of the work reported here was the identification of phosphoproteins and their sites of phosphorylation in WEHI-231 cells. Identification of these proteins is part of the larger AfCS effort to map signaling pathways (6). IMAC coupled to LC-MS/MS has proven to be a powerful method to identify phosphoproteins present in complex mixtures of nonphosphorylated proteins. Of the resins we tested, Ga3+-charged POROS 20 MC and Ga3+-charged BRX-IDA resins resulted in the greatest recovery of the phosphopeptides with the lowest background.
Other groups have utilized the conversion of carboxylic acid groups to methyl esters to reduce nonspecific binding of acidic peptides to IMAC resins (4, 11, 12). We and others (5) have not found that esterification causes a marked enhancement of recovery of phosphopeptides from digests of total cell protein. However, we have found that esterification does enhance the detection and identification of phosphotyrosine peptides from samples previously enriched by immunoaffinity chromatography with anti-phosphotyrosine antibodies.
The proteins identified included known phosphorylated proteins and proteins that are important in B cell signaling. However, Table I is clearly not a complete list of phosphorylated proteins in WEHI-231 cells. A number of known phosphoproteins were not identified in these experiments. Additional enrichment steps will probably be needed to obtain further coverage of the phosphoproteome of these cells. Sixty-five new phosphoproteins were identified in these experiments. These included 54 known proteins and 11 completely novel proteins that have only been inferred from cDNA or genomic sequences. One-third of the total sites (69/193) were proline-directed phosphorylation sites (p-Ser-Pro or p-Thr-Pro), suggesting that many of these proteins are phosphorylated by members of the cyclin-dependent kinase or mitogen-activated protein kinase families. Only one of the sites identified was a phosphorylated tyrosine residue. This result is similar to those reported by others using IMAC methods (4, 11) and probably reflects the low abundance of tyrosine phosphorylation and the fact that a serine/threonine phosphatase inhibitor was used. We have identified significant numbers of p-Tyr-phosphorylated proteins from WEHI-231 cells treated with pervanadate, a tyrosine phosphatase inhibitor, using immunoaffinity purification with anti-p-Tyr antibodies (13).
The information provided in Table I significantly expands the list of potential signaling proteins in B lymphocytes. The identification of novel phosphoproteins should provide new avenues for investigating signaling pathways in these cells. Important issues will be the identification of the protein kinases and phosphatases that act on these sites and identification of factors that lead to changes in their levels of phosphorylation.
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ACKNOWLEDGMENTS |
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We thank Robert Cox, Farah El Mazouni, Kathleen Lyons, and Deepa Sethuraman from the AfCS Protein Chemistry Laboratory for their technical help with this project. We thank Christine Horvath and Robert Hsueh from the AfCS Cell Laboratory for their provision of cells for this work.
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FOOTNOTES |
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Received, November 14, 2003, and in revised form, January 15, 2004.
Published, MCP Papers in Press, January 17, 2003, DOI 10.1074/mpc.D300003-MCP200
1 The abbreviations used are: IMAC, immobilized metal affinity chromatography; AfCS, Alliance for Cellular Signaling; p-Tyr, phosphotyrosine; CLA, calyculin A; LC, liquid chromatography; MS/MS, tandem mass spectroscopy; p-Thr, phosphothreonine. 
* This work was supported by contributions from public and private sources, including the National Institute of General Medical Sciences Glue Grant Initiative (U54 GM062114). A complete listing of AfCS sponsors can be found at www.signaling-gateway.org/aboutus/sponsors.html. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Protein Chemistry Laboratory, Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9196. Tel.: 214-648-2054; Fax: 214-648-5006; E-mail: deirdre.brekken{at}utsouthwestern.edu; web site: www.signaling-gateway.org
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