Phosphoproteome Analysis of HeLa Cells Using Stable Isotope

Labeling with Amino Acids in Cell Culture (SILAC)

Ramars Amanchy, Dario E. Kalume, Akiko Iwahori, Jun Zhong, and Akhilesh Pandey*

McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry and Oncology,Johns Hopkins University, 733 N. Broadway, Baltimore, Maryland 21205

Received May 9, 2005

Identification of phosphorylated proteins remains a difficult task despite technological advances inprotein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) tomass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginineand lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, ofwhich only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 invivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated thephosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breastcancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates withoutprior knowledge of the signaling pathways and can also be applied to profiling to specific kinases incells. Because of its sensitivity and general applicability, our approach will be useful for investigatingsignaling pathways in a global fashion and for using phosphoproteomics for functional annotation ofgenomes.

Keywords: phosphoproteomics • tandem mass spectrometry • SILAC • EGF receptor signaling • tyrosinephosphorylation

Introduction

Reversible phosphorylation of proteins is an importantmechanism for modulating signal transduction pathways.Phosphorylation of proteins is known to regulate enzymaticactivity, subcellular localization, protein-protein interaction,and degradation of proteins. Phosphorylation events relaysignals from extracellular stimuli through interactions that aredependent on phosphorylated residues in specific contexts.1

In eukaryotes, the residues that undergo protein phosphory-lation are serine and threonine, and to a lesser extent, tyrosine.However, the isolation of phosphoproteins from complexmixtures and the determination of phosphorylation sites stillremain a challenge.2,3 Most traditional methods for character-izing the phosphoproteome are limited because of inadequateamounts of proteins and low stoichiometry of phosphorylation.Mass spectrometry has become the technique of choice forphosphoproteome profiling and analysis especially because ofits sensitivity and high-throughput.4,5 Recent developments inmass spectrometry promise to provide novel insights intodynamics of protein activities regulated by post-translationalmodifications.6

A large majority of the available data on tyrosine phospho-rylation events comes from in vitro studies. Although such

experiments help determine whether a molecule could be apotential substrate of a particular kinase, it is experimentallydifficult to confirm if the phosphorylation indeed occurs in vivo.Thus, cell-based strategies are a prerequisite for characteriza-tion of the phosphoproteome to find true protein kinasesubstrates. Oda et al. used stable isotopes (15N) to label yeastproteins for obtaining quantitative information about phos-phorylation events.7 Stable isotope containing amino acids havebeen used by our group previously to obtain similar quantita-tive information about phosphorylation events8 as well as toidentify the targets of protein tyrosine kinases.9 Affinity tech-niques are also used in conjunction with labeling methods toenrich phosphoproteins or phosphopeptides from complexmixtures prior to mass spectrometric analysis. Phosphotyrosineantibodies have been quite successfully used for enrichmentof tyrosine phosphorylated proteins.10,11 Immobilized metalaffinity chromatography (IMAC) has also been used by inves-tigators for purification of serine/threonine as well as tyrosinephosphorylated peptides.12-15

Over 95 protein tyrosine kinases and 107 protein tyrosinephosphatase genes have been described in Homo sapiens.16,17

Although phosphorylation on tyrosine residues of proteinsaccounts for a relatively small fraction of total phosphorylation,it is quite significant in a number of biological processes. Ourobjective was to selectively visualize tyrosine phosphoproteinsusing stable isotope labeling in cell culture (SILAC), followed

* To whom correspondence should be addressed. Tel: (410) 502-6662.Fax: (410) 502-7544. E-mail: pandey@jhmi.edu.

10.1021/pr050134h CCC: $30.25 2005 American Chemical Society Journal of Proteome Research 2005, 4, 1661-1671 1661Published on Web 09/07/2005

by identification using mass spectrometry. We first inducedhyperphosphorylation of proteins on tyrosine residues by aninhibitor of cellular protein tyrosine phosphatases, sodiumpervanadate,18 and subsequently enriched tyrosine phosphop-roteins followed by SDS-PAGE and LC-MS/MS analysis.

Mass spectra from a total of 118 proteins, which exhibitedan enrichment among the proteins derived from pervanadatetreated (heavy isotope containing) cells, were manually ana-lyzed. Forty-five of these proteins have been described to betyrosine phosphorylated while the remaining 73 are not knownto be tyrosine phosphorylated. A total of 42 in vivo tyrosinephosphorylation sites were mapped, including 34 novel sites.A bioinformatics analysis of the phosphorylation sites identifiedin our experiments revealed that most of the tyrosine residueswere not predicted by two commonly used phosphorylationsite prediction programs, Netphos19 and Scansite.20 Importantly,using this approach, we were able to categorize several proteinsof unknown function (e.g., chromosome 3 open reading frame6 (C3ORF6), KIAA0918, and newly identified cDNA from theepidermal differentiation complex-4 (NICE-4) as potentialsignaling molecules.

We validated the phosphorylation status of a subset of theidentified proteins including RNA binding motif protein 4(RBM4), clathrin assembly lymphoid-myeloid leukemia protein(CALM) and transferrin receptor, using antibodies againstendogenous proteins, or, by using epitope-tagged cDNAs in thecase of NICE-4, cytoskeleton-associated protein 1 (CKAP1),breast cancer anti-estrogen resistance 3 (BCAR3), WW domainbinding protein 2, and C3ORF6. As expected, all of theseproteins were highly phosphorylated upon pervanadate treat-ment. In addition, three of these proteins, BCAR3, CKAP1, andC3ORF6, were found to be tyrosine kinase substrates in theEGF receptor signaling pathway as well. Further experimentson the rest of the identified proteins would help unravel theirfunction in protein signaling cascades.

Experimental Section

Chemicals and Reagents. Stable isotope containing aminoacids, 13C9-tyrosine, 13C6-arginine and 13C6-lysine, were pur-chased from Cambridge Isotope Labs (Andover, MA). Completeprotease inhibitor cocktail tablets were purchased from Roche(Indianapolis, IN), sodium orthovanadate from Sigma-AldrichCo (St. Louis, MO), anti-phosphotyrosine antibodies (4G10)agarose-conjugate and streptavidin-agarose beads from UpstateBiotechnology (Lake Placid, NY), antiphosphotyrosine-RC20biotin conjugate from BD transduction laboratories (Lexington,KY) and colloidal Coomassie staining kit from Invitrogen(Carlsbad, CA). Sequencing grade trypsin was purchased fromPromega (Madison, WI).

Stable Isotope Labeling with Amino Acids in Cell Culture.HeLa cells were grown in Dulbecco’s modified Eagle’s mediumcontaining ‘light’ tyrosine, arginine and lysine or ‘heavy’ 13C9-tyrosine, 13C6-arginine and 13C6-lysine supplemented with 10%dialyzed fetal bovine serum plus antibiotics.21 Detailed instruc-tions about this protocol are available at http://www.silac.org.Heavy arginine and lysine were used together in the media.The HeLa cells were adapted to growth in isotope-containingmedium supplemented with dialyzed serum prior to initiatingthese experiments. Pervanadate was freshly prepared beforethe experiment by dissolving equimolar (100 mM) solutions ofsodium orthovanadate and hydrogen peroxide. For each ex-periment, 20 dishes (15 cm) of confluent HeLa cells were usedper condition (untreated or pervanadate-treated). Five percent

of the HeLa cell population grown in light media was ‘spiked’by treating one dish out of 20 dishes with pervanadate. Thecells were serum starved for 2 h before treatment with 1 mMpervanadate for 30 min and were subsequently lysed in amodified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,1mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and1 mM sodium orthovanadate in the presence of proteaseinhibitors) or in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mMNaCl, 1mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycho-late, 1% SDS and 1 mM sodium orthovanadate in the presenceof protease inhibitors).

Cloning and Transfection Experiments. NICE-4(NP•055662), WW domain binding protein 2 (NP•036610),BCAR3 (NP•003558), cytoskeleton-associated protein1 (NP•001272) and chromosome 3 open reading frame 6(NP•777568) were subcloned into a Flag epitope-tagged mam-malian expression vector, pCMVtag4A. 293T cells were trans-fected with cDNAs encoding the above-mentioned proteinsalone using the calcium phosphate transfection method (In-vitrogen, Carlsbad, CA) for the cells to be treated by pervana-date (1 mM) or were cotransfected with EGFR cDNA for thecells to be treated by EGF. The cells were harvested and theimmunoprecipitation with anti-Flag antibody was carried outin modified RIPA buffer.

Immunoprecipitation and Western Blotting. Light andheavy cell lysates were precleared with protein A-agarose,mixed and incubated with 400 µg of 4G10 monoclonal antibod-ies coupled to agarose beads, 75 µg of biotin-conjugated RC20antibody and streptavidin-agarose beads overnight at 4 °C asdescribed earlier.9,21 Precipitated immune complexes were thenwashed three times with lysis buffer and eluted three times with100 mM phenyl phosphate in lysis buffer at 37 °C. The elutedphosphoproteins were dialyzed and resolved by 10% SDS-PAGE. The gels were stained using colloidal Coomassie stain.Western blotting experiments were performed using antiphos-photyrosine antibody (4G10) and reprobing was carried out todetect individual proteins using specific antibodies. For valida-tion of phosphorylation of individual proteins (CALM, RBM4,and transferrin receptor), HeLa cells were grown in 10 cmdishes. One dish was left untreated as control, one was treatedwith EGF for 5 min and the other with 1 mM pervanadate for30 min. Cells were lysed in RIPA buffer. Antibodies againstCALM were kindly provided by Dr. Ernst J. Ungewickell,Hanover Medical School, Hanover, Germany, and antibodiesagainst RBM4 were a gift from Dr. Woan-Yuh Tarn, Instituteof Biomedical Sciences, Taipei, Taiwan.

LC-MS/MS Analysis. The colloidal Coomassie blue stainedprotein bands were excised and digested with trypsin asdescribed previously.21 Briefly, the gel slices were excised, andincubated with trypsin overnight at 37 °C to allow digestion ofproteins after a reduction and alkylation step. After in-geldigestion, the tryptic peptides were extracted. The supernatantfrom the in-gel digestion containing the peptide mixture wasseparated and partially dried down in a vacufuge to ap-proximately 10 µL. The extracted peptide mixture was centri-fuged for 2 min, 12 000 × g at 4 °C to prevent any small piecesof gel from being loaded onto the liquid chromatographysystem.

The peptide mixture was analyzed by reversed phase liquidchromatography tandem mass spectrometry (LC-MS/MS). TheLC system (Agilent 1100 Series, Agilent Technologies, Palo Alto,Ca) was equipped with a well plate sampler, a vacuum degasser,and a capillary pump. Reversed-phase chromatography was

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performed by loading peptides using automated sampler onto2 fused silica capillary columns (OD-360, ID-75 µm) in tandem,a precolumn (5 cm in length) packed with 12 µm C18 ODS-A,(YMC Co, Kyoto, Japan) followed by an analytical column (10cm in length) packed with 5 µm Vydac C18 resin (Nest Group,Southboro, MA). Columns were washed with 95% mobile phaseA (0.4% acetic acid and 0.005% heptafluorobutyric acid, v/v)and 5% mobile phase B (90% acetonitrile, 0.4% acetic acid, and0.005% heptafluorobutyric acid, v/v). The peptides were loadedonto the precolumn (C18 ODS-A) using a linear gradient of 5%mobile phase B. During sample loading the flow rate was keptat 4 µL/min and for peptide separation the flow rate wasdecreased to 250 nL/min. Subsequently, separation of peptideswas carried out in an analytical column using a linear gradientelution from 87% mobile phase A (0.4% acetic acid and 0.005%heptafluorobutyric acid, v/v) to 40% mobile phase B (90%acetonitrile, 0.4% acetic acid and 0.005% heptafluorobutyricacid, v/v) in 34 min. A potential of 2.5 kV was applied to theemitter in the ion source. The spectra were acquired on aMicromass Q-TOF US-API mass spectrometer (Manchester,UK) equipped with an ion source sample introduction systemdesigned by Proxeon Biosystems (Odense, Denmark).

Data Analysis. The acquisition of data was performed usingMassLynx (version 4.0). All spectra were obtained in the positiveion mode. The settings used for the automated data collectionwere as follows: Ion mass window was set to 2.5 Da. The MS/MS to MS switch criteria was set to intensity below a thresholdof 5 counts per second and the MS to MS/MS switch criteriawas set to a threshold corresponding to intensity of 6 countsper second. Scan times of 1 s for MS experiments and 3 s forMS/MS experiments were used and number of MS/MS percycle used was 3 and total cycle time was 10 s with an interscaninterval of 0.1 s. Peptide and protein identification from theMS/MS spectra was carried out as follows. MassLynx wasemployed to generate a peak list (pkl files) from the raw datausing the following parameters: smooth window: 4.00; numberof smooths: 2 (smooth mode: Savitzky Golay); percentage ofpeak height to calculate the centroid spectra: 80%; with nobaseline subtraction. MS/MS spectra were searched the humanRefSeq database22 (build 33) using MASCOT23 (version 1.9) ona Linux cluster. The following settings were used: number oftryptic missed cleavages allowed: 2; peptide window toler-ance: (1.0 Da; and fragment mass tolerance: (0.3 Da. Aminoacid modifications allowed were oxidation of methionine(+16 Da), carbamidomethylcysteine (+57 Da) and phospho-rylation of tyrosine residues (+80 Da). Variable modificationsof 80 Da for tyrosine phosphorylation and 6 and 9 Da for stableisotope containing amino acids, 13C6-Arginine, 13C6-Lysine, and13C9-Tyrosine, respectively, were used. In general, only pep-tides with clear mass spectra with a Mascot score >30 andcontaining a sequence tag of at least four consecutive aminoacids were considered in this study. Peptides with lower scorepresenting a clear tandem mass spectrum were manuallyinterpreted. The intensity ratios were calculated by comparingthe extracted ion chromatograms of the light and heavypeptides.21 The tandem mass spectra were manually verifiedto assign the sequence and phosphorylation sites for allpeptides identified in this study. The phosphorylation sites weremanually verified and assigned after confirmation of a massdifference of 243 Da corresponding to phosphotyrosine residue.Furthermore, presence of an ion marker for phosphotyrosineimmonium ion at m/z 216.043 was also verified. MASCOTresults were parsed using an in-house algorithm developed

using C++ to sort peptides with score >30 for further analysis.Sequence coverage for proteins was calculated based on thesepeptides only. For all purposes, repetition of peptides withsimilar sequence information was eliminated except in situa-tions where the peptide contained variable modifications(oxidation (M), phosphorylation (Y), 13C9-tyrosine, 13C6-arginine,or 13C6-lysine). If a single peptide was observed for a protein,then its N and C termini were verified for tryptic cleavage ends(arginine or lysine). Ion ratios were calculated for all thepeptides with score >30. Further analysis was done on peptideswhich showed increase in the relative intensity (ion ratio).Sequence alignment of human SLITRK family of proteins wasperformed and cladogram was made using an online versionof ClustalW.24

Results and Discussion

SILAC for Differential Labeling of Proteins. SILAC is asimple, in vivo, labeling procedure for investigation of proteindynamics by mass spectrometry and has been successfully usedfor differential labeling of growing cell populations for tacklingthe phosphoproteome and quantitation of phosphorylationevents.8,9,25 HeLa cells were adapted in media containing stableisotope containing amino acids. Two different labeling experi-ments were carried out. In one set of experiments, HeLa cellsgrown in light medium were compared to those grown in 13C6-arginine plus 13C6-lysine. In a second set of experiments, cellsgrown in light medium were compared to those grown in 13C9-tyrosine.9 We labeled the cells with 13C6-arginine and 13C6-lysinebecause it provides a better coverage of labeled peptides.Labeling with 13C9-tyrosine allowed us direct identification ofthe labeled tyrosine containing peptides and also to facilitatedirect identification of sites of phosphorylation on peptidescontaining labeled tyrosine. In both cases, the stable isotopelabeled cells were treated with a tyrosine phosphatase inhibitor,sodium pervanadate which causes hyperphosphorylation oftyrosine residues. The cell populations were harvested and celllysates were mixed and tyrosine phosphorylated proteinsimmunoprecipitated using a combination of monoclonal an-tibodies directed against phosphotyrosine residues as shownin the schematic in Figure 1.

Identification of Tyrosine-Phosphorylated Proteins by LC-MS/MS. Mixing of light and heavy isotope labeled cell lysatesallowed us to compare the profile of proteins in a singleexperiment. In MS/MS spectra, fragmentation patterns gener-ated by light and heavy peptide pairs are identical except forthe expected mass shift of the fragment ions. The peptidescontaining a single 13C9-tyrosine should be heavier by 9 Da,whereas those containing 13C6-arginine or 13C6-lysine residuesshould be heavier by 6 Da. The ratio of the intensity of theheavy versus the light peptides provides information about thedegree of phosphorylation upon pervanadate treatment. Thus,the greater the extent of phosphorylation of a protein, thehigher is its abundance in anti-phosphotyrosine antibodyimmunoprecipitates. Peptide pairs with a little or no increasein intensity indicate that the protein is not different inabundance in the two states being compared. These proteinswere not investigated further as they are likely nonspecificallybound proteins. An increase in heavy/light intensity ratio,indicating an increase in total phosphotyrosine content uponpervanadate treatment, was found in peptides derived from 118proteins by mass spectrometry (Table 1, 2). As an example, weobserved a ratio of 4:1 for a peptide derived from BCAR3 (Figure2 C, D) and 5:1 for a peptide derived from Emerin (Figure 2A,B).

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Seventy-three of the identified proteins have not previouslybeen shown to be phosphorylated on tyrosine residues. Froma total of 13 proteins, we identified 42 phosphorylated tyrosineresidues of which 34 were novel (Table 3, 4). The 73 potentialphosphoproteins that we identified in this study were notknown to be phosphorylated and represent putative tyrosine

kinase substrates. Though a significant fraction of the proteinsidentified seem to be highly abundant proteins, a number ofthem are adapter molecules in signaling pathways, which aregenerally less abundant. Also, we expect that addition ofanother fractionation step would allow identification of an evengreater number of phosphoproteins.

Relative Quantitation of Phosphorylation Events. In addi-tion to identifying phosphorylation sites, mass spectrometryhas been used to deduce the stoichiometry of phosphorylationevents. Absolute quantitative comparison necessitates the useof internal standards,26 which is often impossible while analyz-ing complex mixtures of cell lysates. The isotopically labeledpeptides identified by MASCOT search engine were manuallyverified for the quality of the spectra and sequence. Subse-quently, the partner of the peptide pair was located. Once thepeaks for the peptide pairs were identified, extracted ionchromatograms were generated for each peptide pair and thedegree of increase or decrease in signal was calculated. Wecalculated ion ratios (heavy/light isotope) for multiple peptidescorresponding to 92 proteins from tyrosine labeling experimentand peptide pairs corresponding to 184 proteins from arginineand lysine labeling experiments (Supplementary Table 1 in theSupporting Information). Together from two labeling experi-ments, we have 118 proteins whose intensity was increasedafter pervanadate treatment. On the basis of a literature search,we noted that 45 of these proteins were previously describedto be tyrosine-phosphorylated proteins which fall into variousclasses of molecules (Table 1). To the best of our knowledge,the remaining 73 have not been described to be tyrosine-phosphorylated to date.

Comparison of Arginine Plus Lysine Labeling with TyrosineLabeling. In the case of arginine and lysine labeling, all thepeptides occurring from trypsin cleavage resulted in pairs (lightand heavy) allowing quantitation of phosphorylation events.In the case of tyrosine labeling, only those peptides thatcontained tyrosine resulted in peptide pairs. Thus, the coveragefor the purposes of quantitation was better in arginine pluslysine labeling. The total number of peptide assignments inthis study was 2675, of which 1083 peptides (assigned to 203proteins) were from arginine plus lysine labeling and 1592peptides (assigned to 339 proteins) were from tyrosine labeling.The number of proteins identified from arginine plus lysinelabeling with peptides showing an increase in intensity was 98(39 known + 59 novel) proteins whereas that from tyrosinelabeling was 80 (35 known + 45 novel) proteins. The overlapof proteins from both experiments was 52 (25 known + 27novel) proteins (Figure 5A).

The number of phosphopeptides identified from arginineplus lysine labeling was 37 that provided 41 phosphorylationsites. In contrast, the number of phosphopeptides identifiedfrom tyrosine labeling was 12 that provided 12 phosphorylationsites. The number of overlapping phosphopeptides was 10.Total number of peptide pairs identified from arginine pluslysine labeling was 837 and from tyrosine labeling was 275. Thenumber of peptide pairs showing an increase in the heavy/light ion ratio was 186 from tyrosine labeling and 655 fromarginine plus lysine labeling experiments.

Identification of Tyrosine Phosphorylation Sites. Themass spectra of the peptides for which the phosphorylationsites were assigned by Mascot23 were manually verified for thephosphotyrosine residue. An immonium ion at approximatelym/z 216.043 that is characteristic of phosphorylated tyrosineresidues served as a diagnostic ion for phosphotyrosine con-

Figure 1. Schematic of the strategy used for identification oftyrosine-phosphorylated proteins. Cells were either grown in lightor heavy stable isotope containing media as indicated. The cellpopulation marked as ‘heavy’ was subjected to pervanadatetreatment followed by mixing of cell lysates and immunopre-cipitation of phosphotyrosine containing proteins. The proteinswere resolved by SDS-PAGE as shown and the protein bandsexcised, digested with trypsin, and analyzed by LC-MS/MS. Theintensity ratios of peptide pairs were calculated from the massspectra.

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taining peptides.27 We identified 42 phosphorylation sites(Supplementary Figure 1 in the Supporting Information) in13 proteins out of which 5 are well-known tyrosine phos-phorylated proteins (Table 4). Thirty-four sites out of the42 tyrosine phosphorylation sites identified were novel (Table3). Below, we will briefly discuss some of the interestingproteins where we were able to identify novel phosphorylationsites.

Cortactin: Cortactin is Src substrate28 and has 3 knownphosphorylation sites (Y421, 470 and 486). We identified 3additional phosphotyrosine sites (Y334, 446, 453) in this protein(Figure 3A).

Laminin M: Laminin M/alpha 2 an extracellular protein, isa major component of the basement membrane.29 This rep-resents a rare case of an extracellular protein being phospho-rylated on a tyrosine residue (Y1249) (Figure 3B). The signifi-cance of this phosphorylation is not clear.

MAP1B: Microtubule-associated protein 1B isoform 1 is acytoskeletal protein involved in microtubule assembly and thephosphorylated form of MAP1B plays a crucial role in thedevelopment of the nervous system.30,31 We have mapped 13new tyrosine phosphorylation sites (Y1062, 1762, 1796, 1830,

1870, 1872, 1904, 1905, 1938, 1940, 1957, 1974, 2025) in MAP1B(Figure 3C).

N-Cadherin: N-cadherin is a calcium-dependent transmem-brane adhesion molecule which is known to play a crucial rolein regulating FGF receptor signaling pathway leading to me-tastasis.32 N-Cadherin is known to be a tyrosine-phosphorylatedprotein. We have identified one new tyrosine phosphorylationsite (Y785) in the cytosolic tail of this protein.

Desmoglein 2: Desmoglein 2 (Dsg2) is a calcium-bindingtransmembrane glycoprotein and is a member of desmosomalcadherins.33 Dsg2 is not a known tyrosine phosphorylatedprotein. We have identified a novel tyrosine phosphorylationsite in this protein (Y967).

Catenin, Delta 1: Delta catenin belongs to a group ofstructurally related proteins involved in cell-cell adhesion.It is a known phosphoprotein but the phosphorylation siteson this protein have not yet been identified.34 We haveidentified 7 new phosphorylation sites in this protein (Y96,174, 217, 228, 257, 302, 859), which could play a role in celladhesion.

Aspartate Aminotransferase 2: This is a pyridoxal phosphate-dependent enzyme localized to inner-membrane of mitochon-

Table 1. List of Novel Potential Tyrosine Phosphorylated Proteins Identified in This Study

proteinRefSeq

accession proteinRefSeq

accession

Cellular Communication and Signal Transduction Nucleic Acid Synthesis and Processing1 BAI1-associated protein 2 isoform 3 NP•006331 44 Cleavage and polyadenylation specific factor 5 NP•0089372 Brain-enriched guanylate kinase-associated protein NP•065887 45 LIM domain only 7 NP•0053493 Breast cancer antiestrogen resistance 3 NP•003558 46 Poly(rC)-binding protein 2, isoform b NP•1143664 Caveolin-3 NP•001225 47 Polymerase I and transcript release factor NP•0363645 Cytoplasmic FMR1 interacting protein 1 NP•055423 48 Pre-mRNA cleavage factor I NP•0790876 Notch 2 preproprotein NP•077719 49 RNA binding motif protein 4 NP•0028877 Palmdelphin NP•060204 50 SH2 domain binding protein 1 NP•0554488 Partitioning-defective protein 3 homolog NP•062565 Energy and Metabolism9 Pleckstrin homology-like domain, family B, member 2 NP•665696 51 Acetyl-Coenzyme A carboxylase alpha NP•00065510 Programmed cell death 6 interacting protein NP•037506 52 Aspartate aminotransferase 2 NP•00207111 Prohibitin NP•002625 53 ATP synthase, H+ transporting, beta NP•00167712 Rho guanine nucleotide exchange factor 7 isoform a NP•003890 54 High-glucose-regulated protein 8 NP•05734213 Sorcin isoform a NP•003121 55 Inosine monophosphate dehydrogenase 2 NP•00087514 TRK-fused gene NP•006061 56 Phosphogluconate dehydrogenase NP•00262215 Tumor necrosis factor type 1 receptor associated protein NP•057376 57 Propionyl Coenzyme A carboxylase, beta polypeptide NP•000523

Cellular Organization 58 Propionyl-Coenzyme A carboxylase, alpha polypeptide NP•00027316 Actin, gamma 1 propeptide NP•001605 59 Protein-L-isoaspartate O-methyltransferase NP•00538017 Actin-binding LIM protein 1 isoform s NP•006711 60 Pyruvate carboxylase precursor NP•00091118 Actin-related protein 2 NP•005713 Storage and Transport19 Actin-related protein 3 NP•005712 61 Clathrin assembly lymphoid-myeloid leukemia protein NP•00909720 Calponin-3 NP•001830 62 Phosphate carrier precursor, isoform 1b NP•00262621 Destrin (actin depolymerizing factor) NP•006861 63 SEC23A NP•00635522 Echinoderm microtubule associated protein like 4 NP•061936 64 SEC24C NP•00631423 Emerin NP•000108 65 Solute carrier family 25 (adenine nucleotide translocator) NP•00114224 Epiplakin 1 NP•112598 66 Transferrin receptor/CD71 NP•00322525 Kindlin 1 NP•060141 67 Voltage-dependent anion channel 2 NP•00336626 Laminin M NP•000417 Immunity27 LIM and SH3 protein 1 NP•006139 68 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3 NP•00134728 Myosin IE NP•004989 69 p53 inducible protein NP•05519129 Plakophilin 4 isoform a NP•003619 Unclassified30 Plectin 1 isoform 1 NP•000436 70 Chromosome 3 open reading frame 6 NP•77756831 Similar to Myosin heavy chain, nonmuscle type B XP•290747 71 KIAA0918 NP•05638232 Spectrin, beta, nonerythrocytic 1 isoform 1 NP•003119 72 NICE-4 protein NP•05566233 Tight junction protein 2 NP•004808 73 Similar to KIAA0310 protein XP•08845934 Tubulin, alpha, 6 NP•11609335 WAS protein family, member 2 NP•008921

Protein Synthesis Processing and Protein Fate36 Actin, beta NP•00109237 Cytoskeleton-associated protein 1 NP•00127238 Eukaryotic translation elongation factor 2 NP•00195239 Eukaryotic translation initiation factor 4B NP•00140840 HBS1-like NP•00661141 Ubiquitin and ribosomal protein S27a NP•00294542 Ubiquitin associated protein 2 isoform 1 NP•06091943 WW domain binding protein 2 NP•036610

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dria that plays a role in glutamate metabolism, urea andtricarboxylic acid cycles.35 It is not known to be phosphorylatedon tyrosine residues. We have identified a phosphorylation site(Y401) in this enzyme.

Emerin: Emerin is a serine-rich nuclear membrane proteinassociated with nuclear lamina.36 Emerin is considered to be aserine phosphorylated protein and we have added a novelfetature to this protein through the mapping of 5 tyrosinephosphorylation sites (Y59, 74, 85, 161, 167).

Valosin-Containing Protein: Valosin-containing protein (VCP)is yeast Cdc48p homologue37 and is a known tyrosine phos-phorylated protein. We have identified a phosphotyrosineresidue in its C-terminus (Y805).

KIAA0918: This protein was originally isolated as a cDNAfragment from a brain library.38 It belongs to the human SLITRK

family of transmembrane signaling molecules. Our analy-sis identified a phosphorylated tyrosine residue (Y833) in thecytoplasmic domain of this transmembrane protein (Figure3, Panel D). Like the cytoplasmic residues found in severalother transmembrane receptors, it is likely that this tyrosinephosphorylation site is involved in signaling by this novelreceptor.

Experimental Validation of Tyrosine Phosphorylation andFunctional Elucidation of Identified Proteins in EGF ReceptorSignaling. As commercial antibodies were not available forvalidation of all of the potential phosphoproteins identified, itwas difficult to confirm their tyrosine phosphorylation usingWestern blotting. To validate our findings, we selected a subsetof proteins (RBM4, CALM, and transferrin receptor) againstwhich immunoprecipitating antibodies were available andwhich were not previously shown to be phosphorylated. Wesubjected these proteins to immunoprecipitation using specificantibodies followed by Western blotting with anti-phosphoty-rosine antibodies (Figure 4A). We also analyzed a subset ofproteins against which no antibodies are available. For thispurpose, we first obtained full-length cDNA clones and gener-ated Flag epitope-tagged versions of NICE-4, WW domainbinding protein 2, BCAR3, CKAP1, and C3ORF6. We transfectedthese Flag tagged cDNAs into 293T cells and treated them withpervanadate (Figure 4B). As these proteins were identified fromHeLa cells which express EGF receptors, we also chose toinvestigate the functional relevance of these novel phosphop-roteins in EGF receptor signaling by either stimulating HeLacells with EGF or by coexpressing EGF receptor with theseproteins in 293T cells (Figure 4B). As expected, all the proteinsthat were tested, exhibited increased tyrosine phosphorylationupon pervanadate treatment. Additionally, BCAR3, CKAP1, andC3ORF6 were phosphorylated upon EGF treatment therebyimplicating them as molecules that are involved in EGFsignaling pathway.

RBM4 is a protein with RNA recognition motifs and zincfinger domain characteristic of RNA binding proteins.39,40 Wehave identified this protein to be tyrosine phosphorylated(Figure 4A). RBM4 has a proline rich segment in its C-terminuswhich has known ligands containing protein interaction do-mains such as WW41 and SH3 domains.42 Transferrin receptoris a receptor for transferrin whose main function is to serve asa sensor for the iron in serum bound to transferrin and takingup the iron by endocytosis.43 Transferrin receptor is not knownto be a phosphoprotein. We have established transferrinreceptor as a tyrosine phosphorylated protein (Figure 4A). Wealso identified CALM, a phosphatidylinositol and clathrin heavychain binding protein to be phosphorylated (Figure 4A). AC-terminal segment of CALM probably interacts with proteinsin the plasma membrane, possibly contributing to the regula-tion of the endocytotic activity, mediated by phosphoinositolpathways.44

From our experiments, we were able to implicate 3 mol-ecules, BCAR3, CKAP1, and C3ORF6, as novel tyrosine kinasesubstrates in the EGF receptor signaling pathway (Figure 4B).BCAR3, contains a src homology 2 (SH2) domain, a hallmarkof cellular tyrosine kinase signaling molecules and was initiallydiscovered in a search for genes involved in antiestrogenresistance of human breast cancer cells, which makes it a veryinteresting candidate because of the involvement of the EGFreceptor family in breast cancer progression.45 Cytoskeletonassociated protein 1 (CKAP1), contains CAP-GLY domain whichis a glycine region highly conserved among several cytoskel-

Table 2. List of Known Tyrosine Phosphorylated ProteinsIdentified in This Study

proteinRefSeq

accession

Cellular Communication and Signal Transduction1 Annexin VII isoform 1 NP•0011472 Annexin XI NP•0011483 Breast cancer anti-estrogen resistance 1 (p130CAS) NP•0553824 Catenin, delta 1 NP•0013225 Ephrin receptor EphB4 NP•0044356 ERBB2 interacting protein isoform 2 NP•0611657 G protein-coupled receptor kinase interactor 1 NP•0547498 GAP-associated tyrosine phosphoprotein NP•0065509 Hematopoietic cell-specific Lyn substrate 1 NP•00532610 Hepatocyte growth factor-regulated tyrosine

kinase substrateNP•004703

11 Integrin, beta 4 isoform 1 NP•00020412 N-cadherin NP•00178313 NCK-associated protein 1 NP•03846414 Plakoglobin NP•00222115 Proline-serine-threonine phosphatase

interacting protein 2NP•077748

16 PTK2 protein tyrosine kinase 2 isoform a NP•72256017 Signal transducing adaptor molecule 1 NP•00346418 Signal transducing adaptor molecule 2 NP•00583419 Src homology 3 domain-containing protein

HIP-55NP•054782

20 Target of Myb1-like 1 NP•00547721 Viral oncogene yes-1 homologue 1 NP•005424

Cellular Organization22 Actinin, alpha 1 NP•00109323 Actinin, alpha 4 NP•00491524 Clathrin, heavy chain 1 NP•00485025 Cofilin 1 NP•00549826 Cortactin, isoform a NP•00522227 Cortactin, isoform b NP•61263228 Desmoglein 2 NP•00193429 Filamin 1 NP•00144730 Microtubule-associated protein 1B isoform 1 NP•00590031 Myosin, heavy polypeptide 9, nonmuscle NP•00246432 Paxillin NP•00285033 Spectrin, alpha, nonerythrocytic 1 NP•00311834 Tight junction protein 1, isoform a NP•00324835 Tight junction protein 1, isoform b NP•78329736 Tubulin, alpha, ubiquitous NP•00607337 Tubulin, beta, 2 NP•00607938 Villin 2 NP•00337039 Vimentin NP•003371

Protein Synthesis Processing and Protein Fate40 Valosin-containing protein NP•009057

Nucleic Acid Synthesis and Processing41 Ewing sarcoma breakpoint region 1 NP•00523442 Heterogeneous nuclear ribonucleoprotein K NP•11255243 NS1-associated protein 1 NP•006363

Energy and Metabolism44 Lactate dehydrogenase A NP•005557

Storage and Transport45 Nephrin related gene NP•060710

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eton-associated proteins.46 We have also identified CKAP1 tobe tyrosine phosphorylated in response to EGF stimulation(Figure 4B). Finally, tyrosine phosphorylation of C3ORF6 uponactivation by EGF indicates that C3ORF6 is likely to be anadapter molecule in the EGF signaling pathway (Figure 4B).Progress in finding tyrosine kinase substrates in signalingpathways is limited in part by a need for experimental ap-proaches that can isolate and identify tyrosine-phosphorylatedpeptides in large numbers. Current proteomic approachesgenerally reveal only small numbers of tyrosine phosphoryla-tion sites. Direct analysis of EGF receptor signaling pathway25,47

and FGF receptor signaling pathway48 have been carried out.Further experiments need to be carried out to dissect the roleof these three molecules in the EGF receptor signaling cascade.While this manuscript was in preparation Hinsby et al. used asimilar strategy to investigate phosphotyrosine containingproteins in FGF receptor signaling pathway.48 Two other globalapproaches that have recently been published include analysisof the yeast pheromone signaling49 pathway using SILAC andimmunoaffinity profiling of cancer cells in pervanadate-treatedcells using phosphospecific antibodies.50 Nevertheless, ourresults are distinctive and the tyrosine phosphorylation sites

that we identified have not been reported in these studies againindicating that complementary approaches are necessary fora comprehensive analysis of the phosphoproteome.

Bioinformatics Analysis of Tyrosine Phosphoproteins andPhosphorylation Sites. Gene ontology (GO) terms have beenwidely used to categorize and classify proteins based onbiological process and cellular component to assign molecularfunctions to proteins.51 A functional annotation of the proteinsidentified in this study was carried out by categorizing theproteins into divisions based on biological processes (Figure5B). Both known and novel proteins were categorized andclassified on the basis of GO biological process terms (Tables1, 2). Out of the 118 proteins identified, ∼60% of the proteinswere cytoskeletal and signaling proteins. On the basis of thepublished literature, one would expect signaling proteins andproteins associated with the cytoskeleton to be tyrosine-phosphorylated. Surprisingly, however, our results also revealedeight key metabolic enzymes to be probable substrates oftyrosine kinases although lactate dehydrogenase was the onlyone that was previously known to be phosphorylated. We wereable to identify a phosphorylation site in the metabolic enzyme,aspartate aminotransferase 2. We found several proteins with

Figure 2. Identification of tyrosine phosphorylated proteins by mass spectrometry. (A) A mass spectrum showing a peptide pair(containing 12C9 and 13C9-tyrosine, respectively) differing by 9 Da and exhibiting a ratio of 1:5. (B) Product ion mass spectrum (MS/MS)of the doubly charged ion at m/z 547.78 (from panel A). The spectrum corresponds to the peptide sequence (with 13C9-tyrosine), IFEYETQR,derived from the protein, emerin. (C) A mass spectrum showing a peptide pair (containing 12C6 and 13C6-arginine, respectively) differingby 6 Da and exhibiting a ratio of 1:4. (D) Product ion mass spectrum (MS/MS) of the doubly charged ion at m/z 598.80 (from panel C)which corresponds to the peptide sequence (with 13C6-arginine), GGSGATLEDLDR, derived from the protein, breast cancer antiestrogenresistance 3 (BCAR3).

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a functional role in nucleic acid and protein synthesis andprocessing to be potential phosphoproteins. We have experi-mentally proven the tyrosine phosphorylation of an RBM4, anovel conserved zinc finger-containing RNA-binding protein,which is found to be involved in RNA processing39 and of astorage and transport protein, CALM (Figure 4A). We haveidentified several phosphotyrosine residues in MAP1B. Like-wise, our results also revealed 7 new phosphorylated tyrosineresidues in delta catenin and 5 in emerin. Investigation of thesignificance of these modifications will help in further under-standing the role of these proteins.

For all the proteins where we were able to map tyrosinephosphorylation sites, an analysis was carried out using twopopular phosphorylation prediction programs, Netphos19 andScansite20 (Table 3, 4). In searches using Netphos, an outputscore of 0.5 was used as cutoff to ensure that the site was abona fide phosphorylation site. We used a more sensitiveapproach using Scansite by selecting a low stringency output.Remarkably, as we have reported earlier,47 these programs failedto predict most of the novel tyrosine-phosphorylated sites.

There are a large number of hypothetical proteins in proteindatabase for which functions cannot be easily assigned.However, if they are identified as tyrosine phosphorylatedproteins, it is possible to test their specific role more systemati-cally in various tyrosine kinase signaling pathways. In thisstudy, we have identified 4 uncharacterized proteins, whichexisted only as cDNA sequences in database, as phosphoty-rosine containing proteins. The KIAA0918 protein has a signalpeptide, several LRR repeats and a transmembrane domain(Figure 6A). An alignment with related protein shows that thisprotein belongs to the human SLITRK family of signalingproteins (Figure 6B). Aruga et al. have previously pointed outthe conservation of several tyrosine residues in the SLITRKfamily although there is no published evidence for phospho-rylation for any of these sites.52 We identified Y833, which islocated in the cytosolic tail of SLITRK5 and conserved in somemembers of this family, as a phosphorylation site.53 Thesimilarity between C-terminal domain of SLITRK family ofproteins and trk neurotrophin receptor suggests a possibleinvolvement in PLCγ-mediated signaling53 and could involve

Table 3. List of Novel Tyrosine Phosphorylation Sites Identified in This Study

protein Refseq

Identified phosphopeptide

Sequence netphos scansite

Microtubule-associatedprotein 1B isoform 1

NP•005900 DMSLpYASLTSEK yes yes

TPEEGGYSpYDISEK yes yesTPEDGDpYSpYEIIEK yes yesESSPLpYSPTFSDSTSAVK no noITSFPESEGYSpYETSTK yes noAAEAGGAEEQpYGFLTTPTK yes yesTTSPPEVSGYSpYEKTER yes yesTSDVGGpYpYYEKIER no noTATCHSSSSPPIDAASAEPpYGFR yes noTPGDFSpYApYQKPEETTR no noTPGDFSpYAYQKPEETTR no yes

N-cadherin 1 NP•001783 YDEEGGGEEDQDpYDLSQLQQPDTVEPDAIKPVGIR yes noAspartate aminotransferase 2 NP•002071 EFSIpYMTK yes noDesmoglein 2 NP•001934 VpYAPASTLVDQPYANEGTVVVTER yes noLaminin M NP•000417 KLMApYGGK no noCatenin, delta 1 NP•001322 LNGPQDHSHLLpYSTIPR yes no

QDVpYGPQPQVR no noSQSSHSpYDDSTLPLIDR yes noHYEDGYPGGSDNpYGSLSR yes noHpYEDGYPGGSDNpYGSLSR yes noTVQPVAMGPDGLPVDASSVSNNpYIQTLGR no noSMGYDDLDYGMMSDpYGTAR yes no

Cortactin, isoform a NP•005222 NASTFEDVTQVSSApYQK no noGPVSGTEPEPVpYSMEAADYR yes noGPVSGTEPEPVpYSMEAADpYR no no

Emerin NP•000108 KEDALLpYQSK yes noDSApYQSITHpYRPVSASR 1-yes noIFEYETQRRLSPPSSSAASSpYSFSDLNSTR yes noGDADMpYDLPKKEDALLYQSK yes no

KIAA0918/SLITRK5 NP•056382 SPApYSVSTIEPR yes noValosin-containing protein NP•009057 FPSGNQGGAGPSQGSGGGTGGSVYTEDNDDDLpYG yes noHepatocyte growth factor-regulated tyrosine kinase substrate

NP•004703 AEPMPSASSAPPASSLpYSSPVNSSAPLAEDIDPELAR yes no

Table 4. List of Known Tyrosine Phosphorylation Sites Identified in This Study

protein Refseq identified phosphopeptide sequence netphos scansite

Breast cancer anti-estrogenresistance 1 (p130CAS)

NP•055382 AQQGLpYQVPGPSPQFQSPPAK no no

RPGPGTLpYDVPR yes yesHLLAPGPQDIpYDVPPVR yes yesVGQGpYVpYEAAQPEQDEYDIPR yes noVGQGYVYEAAQPEQDEpYDIPR yes no

Paxillin NP•002850 FIHQQPQSSSPVpYGSSAK yes noVGEEEHVpYSFPNK yes yes

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binding of phosphorylated SLITRK5 to SH2 and/or PTB domaincontaining proteins. C3ORF6 was originally identified by

computational methods followed by experimental validationby PCR amplification.54 It contains a coiled-coil domain and is

Figure 4. Experimental validation of tyrosine phosphorylation status by Western blotting. HeLa cells were treated with 1 mM pervanadatefor 30 min or 10 ng/mL EGF for 5 min. (A) Antibodies against RNA binding motif protein 4 (RBM4), transferrin receptor and clathrinassembly lymphoid-myeloid leukemia protein (CALM) were used for immunoprecipitation followed by SDS-PAGE and Western blotting.The blots were first probed with anti-phosphotyrosine antibody and then stripped and reprobed with antibodies against the individualproteins as shown. (B) 293T cells were transfected with Flag-tagged cDNAs of BCAR3 (breast cancer anti-estrogen resistance 3), CKAP1(cytoskeleton-associated protein 1), WW domain binding protein 2 (WWBP2), chromosome 3 open reading frame 6 (C3ORF6) andNICE-4, and were treated with 1 mM pervanadate for 30 min or cotransfected with EGF receptor and stimulated with EGF. The expressedproteins were immunoprecipitated using anti-Flag antibody, followed by SDS-PAGE and Western blotting. The blots were probed withanti-phosphotyrosine antibody followed by reprobing with anti-Flag as shown.

Figure 3. Mapping of tyrosine phosphorylation sites. MS/MS spectra of four different tyrosine phosphorylated peptides containingeither 13C6-arginine or 13C6-lysine. (A) The peptide sequence, GPVSGTEPEPVpYSMEAADpYR, is derived from cortactin and containstwo novel tyrosine phosphorylation sites as indicated, Y446 and Y453. (B) The peptide sequence KLMApYGGK, is derived from lamininM. (C) The peptide sequence TPEEGGYSpYDISEK, is derived from microtubule-associated protein 1B isoform 1 (MAP1B). (D) The peptidesequence SPApYSVSTIEPR, is derived from a novel protein (KIAA0918/SLITRK5). pY denotes phosphorylated tyrosine residues.

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well conserved across species.54 We have validated its tyrosinephosphorylation status both in response to pervanadate treat-ment and in the EGF signaling pathway. NICE-4, identifiedas part of human epidermal differentiation complex,55 has aubiquitin associated domain and we have demonstrated itsphosphorylation upon pervanadate treatment.

Conclusions

Our results demonstrate the utility of the SILAC method forin vivo differential labeling of proteins for the identification oftyrosine-phosphorylated proteins and localization of phospho-rylation sites. In addition to identification of known tyrosinephosphorylation sites on p130CAS, Hrs and paxillin, we haveidentified novel sites on MAP1B, N-cadherin, aspartate ami-notransferase 2, desmoglein 2, laminin M, catenin delta 1,cortactin, emerin and valosin-containing protein. Nevertheless,we have missed a number of known phosphorylation proteinsperhaps owing to their lack of expression in HeLa cells, lowabundance levels and/or lack of adequate enrichment usingour methods. We have verified 3 novel proteins as downstreamintermediates in the EGF receptor signaling pathway. Weidentified novel hypothetical proteins KIAA0918 and C3ORF6and confirmed that they are phosphoproteins. A focus on theseindividual proteins would reveal the specific role of theseproteins and novel sites in signaling cascades and the signifi-cance of tyrosine phosphorylation in the regulation of theirfunction. Mass spectrometry followed by experimental valida-tion of phosphorylation and verification of the sites of phos-phorylation and their significance would provide biologicalinformation. Because of its sensitivity and selectivity, thisstrategy will be useful in proteomic approaches to investigatetyrosine as well as serine/threonine phosphorylation in signal-ing. Finally, modifications of this strategy could be used toinvestigate protein-protein interactions and multiprotein com-plexes that are formed in response to specific stimuli.

Acknowledgment. This work was supported by NHLBIContract HV-28180 from the National Institutes of Health andby a Career Development Award from the Breast Cancer SPORE(CA 88843) at the Sidney Kimmel Comprehensive CancerCenter at Johns Hopkins. We thank Pratap Vedula for hisassistance with programming.

Figure 5. Functional annotation of the tyrosine phosphopro-teome. (A) A comparison of the two different labeling approachesand the overlap of proteins and phosphorylation sites from thetwo experiments. (B) The pie chart shows the distribution ofbiological processes to the known and potential phosphoproteinsidentified in this study using Gene Ontology terms.

Figure 6. Functional annotation of an uncharacterized/novel protein, KIAA0918/SLITRK5. (A) The domain architecture of a novel humanprotein KIAA0918/SLITRK5 shows a signal peptide, numerous leucine-rich repeats (LRR), LRR C and N-terminal domains (LRR CT andLRR NT) and a transmembrane domain. (B) A cladogram of the human SLITRK family of proteins.

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Supporting Information Available: Calculated ionratios (heavy/light isotope) for multiple peptides correspondingto 92 proteins from tyrosine labeling experiment and peptidepairs corresponding to 184 proteins from arginine and lysinelabeling experiments (Supplementary Table 1); 42 identifiedphosphorylation sites (Supplementary Figure 1). This materialis available free of charge via the Internet at http://pubs.acs.org.

References

(1) Pawson, T.; Nash, P. Science 2003, 300, 445-452.(2) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-

382.(3) Kalume, D. E.; Molina, H.; Pandey, A. Curr. Opin. Chem. Biol.

2003, 7, 64-69.(4) Zhang, H.; Yan, W.; Aebersold, R. Curr. Opin. Chem. Biol. 2004,

8, 66-75.(5) Yates, J. R. 3rd Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 297-

316.(6) Jensen, O. N. Curr. Opin. Chem. Biol. 2004, 8, 33-41.(7) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc.

Natl. Acad. Sci. U.S.A. 1999, 96, 6591-6596.(8) Ibarrola, N.; Kalume, D. E.; Gronborg, M.; Iwahori, A.; Pandey,

A. Anal. Chem. 2003, 75, 6043-6049.(9) Ibarrola, N.; Molina, H.; Iwahori, A.; Pandey, A. J. Biol. Chem.

2004, 279, 15805-15813.(10) Pandey, A.; Podtelejnikov, A. V.; Blagoev, B.; Bustelo, X. R.; Mann,

M.; Lodish, H. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 179-184.(11) Soskic, V.; Gorlach, M.; Poznanovic, S.; Boehmer, F. D.; Godovac-

Zimmermann, J. Biochemistry 1999, 38, 1757-1764.(12) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1,

207-222.(13) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892.(14) Loughrey Chen, S.; Huddleston, M. J.; Shou, W.; Deshaies, R. J.;

Annan, R. S.; Carr, S. A. Mol. Cell Proteomics 2002, 1, 186-196.(15) Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q.

T.; Ericson, C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.;Peters, E. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 443-448.

(16) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,S. Science 2002, 298, 1912-1934.

(17) Alonso, A.; Sasin, J.; Bottini, N.; Friedberg, I.; Osterman, A.; Godzik,A.; Hunter, T.; Dixon, J.; Mustelin, T. Cell 2004, 117, 699-711.

(18) Huyer, G.; Liu, S.; Kelly, J.; Moffat, J.; Payette, P.; Kennedy, B.;Tsaprailis, G.; Gresser, M. J.; Ramachandran, C. J. Biol. Chem.1997, 272, 843-851.

(19) Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999, 294, 1351-1362.

(20) Yaffe, M. B.; Leparc, G. G.; Lai, J.; Obata, T.; Volinia, S.; Cantley,L. C. Nat. Biotechnol. 2001, 19, 348-353.

(21) Amanchy, R.; Kalume, D. E.; Pandey, A. Sci. STKE 2005, 2005,pl2.

(22) Pruitt, K. D.; Maglott, D. R. Nucleic Acids Res. 2001, 29, 137-140.(23) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S.

Electrophoresis 1999, 20, 3551-3567.(24) Pearson, W. R.; Lipman, D. J. Proc. Natl. Acad. Sci. U.S.A. 1988,

85, 2444-2448.(25) Blagoev, B.; Kratchmarova, I.; Ong, S. E.; Nielsen, M.; Foster, L.

J.; Mann, M. Nat. Biotechnol. 2003, 21, 315-318.(26) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P.

Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940-6945.(27) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal.

Chem. 2001, 73, 1440-1448.

(28) Kapus, A.; Szaszi, K.; Sun, J.; Rizoli, S.; Rotstein, O. D. J. Biol. Chem.1999, 274, 8093-8102.

(29) Dowgiert, J.; Sosne, G.; Kurpakus-Wheater, M. Cell Prolif. 2004,37, 161-175.

(30) Avila, J.; Ulloa, L.; Diez-Guerra, J.; Diaz-Nido, J. Cell Biol. Int. 1994,18, 309-314.

(31) Pedrotti, B.; Ulloa, L.; Avila, J.; Islam, K. Biochemistry 1996, 35,3016-3023.

(32) Suyama, K.; Shapiro, I.; Guttman, M.; Hazan, R. B. Cancer Cell2002, 2, 301-314.

(33) Schafer, S.; Stumpp, S.; Franke, W. W. Differentiation 1996, 60,99-108.

(34) Martinez, M. C.; Ochiishi, T.; Majewski, M.; Kosik, K. S. J. CellBiol. 2003, 162, 99-111.

(35) Piva, T. J.; McEvoy-Bowe, E. J. Cell Biochem. 1998, 68, 213-225.(36) Bengtsson, L.; Wilson, K. L. Curr. Opin. Cell Biol. 2004, 16, 73-

79.(37) Kobayashi, T.; Tanaka, K.; Inoue, K.; Kakizuka, A. J. Biol. Chem.

2002, 277, 47358-47365.(38) Nagase, T.; Ishikawa, K.; Suyama, M.; Kikuno, R.; Hirosawa, M.;

Miyajima, N.; Tanaka, A.; Kotani, H.; Nomura, N.; Ohara, O. DNARes. 1998, 5, 355-364.

(39) Jackson, F. R.; Banfi, S.; Guffanti, A.; Rossi, E. Genomics 1997,41, 444-452.

(40) Lai, M. C.; Kuo, H. W.; Chang, W. C.; Tarn, W. Y. Embo. J. 2003,22, 1359-1369.

(41) Chen, H. I.; Sudol, M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7819-7823.

(42) Ren, R.; Mayer, B. J.; Cicchetti, P.; Baltimore, D. Science 1993,259, 1157-1161.

(43) Cheng, Y.; Zak, O.; Aisen, P.; Harrison, S. C.; Walz, T. Cell 2004,116, 565-576.

(44) Kalthoff, C.; Alves, J.; Urbanke, C.; Knorr, R.; Ungewickell, E. J. J.Biol. Chem. 2002, 277, 8209-8216.

(45) Slamon, D. J.; Godolphin, W.; Jones, L. A.; Holt, J. A.; Wong, S.G.; Keith, D. E.; Levin, W. J.; Stuart, S. G.; Udove, J.; Ullrich, A. etal. Science 1989, 244, 707-712.

(46) Watanabe, T. K.; Shimizu, F.; Nagata, M.; Kawai, A.; Fujiwara, T.;Nakamura, Y.; Takahashi, E.; Hirai, Y. Cytogenet. Cell Genet 1996,72, 208-211.

(47) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. J. Biol.Chem. 2002, 277, 1031-1039.

(48) Hinsby, A. M.; Olsen, J. V.; Mann, M. J. Biol. Chem. 2004, 279,46438-46447.

(49) Gruhler, A.; Olsen, J. V.; Mohammed, S.; Mortensen, P.; Faerge-man, N. J.; Mann, M.; Jensen, O. N. Mol. Cell Proteomics 2005, 4,310-327.

(50) Rush, J.; Moritz, A.; Lee, K. A.; Guo, A.; Goss, V. L.; Spek, E. J.;Zhang, H.; Zha, X. M.; Polakiewicz, R. D.; Comb, M. J. Nat.Biotechnol. 2005, 23, 94-101.

(51) Ashburner, M.; Ball, C. A.; Blake, J. A.; Botstein, D.; Butler, H.;Cherry, J. M.; Davis, A. P.; Dolinski, K.; Dwight, S. S.; Eppig, J. T.;Harris, M. A.; Hill, D. P.; Issel-Tarver, L.; Kasarskis, A.; Lewis, S.;Matese, J. C.; Richardson, J. E.; Ringwald, M.; Rubin, G. M.;Sherlock, G. Nat. Genet. 2000, 25, 25-29.

(52) Aruga, J.; Yokota, N.; Mikoshiba, K. Gene 2003, 315, 87-94.(53) Aruga, J.; Mikoshiba, K. Mol. Cell Neurosci. 2003, 24, 117-129.(54) Vazza, G.; Picelli, S.; Bozzato, A.; Mostacciuolo, M. L. Gene 2003,

314, 113-120.(55) Marenholz, I.; Zirra, M.; Fischer, D. F.; Backendorf, C.; Ziegler,

A.; Mischke, D. Genome Res. 2001, 11, 341-355.

PR050134H

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