Tandem Immunoprecipitation of Phosphotyrosine-Mass

Tandem Immunoprecipitation of Phosphotyrosine-Mass Spectrometry (TIPY-MS) Indicates C19ORF19 Becomes Tyrosine-Phosphorylated and Associated with Activated Epidermal Growth Factor Receptor Jiefei Tong,†,‡ Paul Taylor,†,‡ Eleonora Jovceva,†,‡ Jonathan R. St-Germain,†,‡ Lily L. Jin,†,‡ Ana Nikolic,†,‡ Xiaoping Gu,†,‡ Zhi Hua Li,‡,§ Suzanne Trudel,‡,§ and Michael F. Moran*,†,‡ Program in Molecular Structure and Function, Hospital for Sick Children, Department of Molecular Genetics, and Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada, McLaughlin Centre for Molecular Medicine, Toronto, Canada, and Hematology-Oncology, Princess Margaret Hospital, Toronto, Canada Received October 3, 2007

To identify phosphotyrosine (pY) sites in the epidermal growth factor receptor (EGFR) network, a tandem immunoprecipitation-mass spectrometry method (TIPY-MS) was applied wherein protease-digested EGFR immune complexes were extracted with anti-pY after Rush et al. (Nat. Biotech. 2005, 23, 94) and analyzed by LC-MS/MS. New pY sites in the pathway were found, including SOS1 Y1065, SOS2 Y1275, CBL-B Y889, and in the EGFR regulatory protein Mig-6 Y458. The novel human C19orf19 gene product was found EGFR-associated and phosphorylated at 5 tyrosines in response to EGFR activation and, therefore, represents a new component of the EGFR signaling network. Keywords: C19orf19 • EGFR • epidermal growth factor • mass spectrometry • phosphotyrosine • phospho-proteomics • protein–protein interaction

Introduction

individual EGFR-directed data sets, as with global/broad interaction and phosphorylation data sets, is typically only partial. To some extent, this is due to the sampling nature of MS methods, but also indicates that our knowledge of EGFR phospho-proteomes and interactomes is incomplete.29,30

Reversible protein–protein interactions and phosphorylation are major mechanisms of cell regulation, and fundamental to the function of protein-tyrosine kinases such as the epidermal growth factor receptor (EGFR). In metazoans, the sequence-specific binding of SH2 and PTB domains to phosphotyrosine (pY) sites in receptor tyrosine kinases (RTKs) such as the EGFR exemplifies how protein phosphorylation and protein–protein interactions functionally integrate into intracellular signaling networks.1 Largescale mass spectrometry (MS)-based studies of protein phosphorylation2–10 and protein complexes11–15 have, in aggregate, revealed thousands of protein phosphorylation sites and protein– protein interactions, respectively. Protein microarray16 and MSbased systematic approaches17 aimed at defining the phosphorylation-dependent interactome of the EGFR, and studies of EGFR-associated protein phosphorylations,6,7,18–26 have identified many new components of the EGFR network. Another integrated approach to define EGFR pathway components is the MS analysis of proteins co-immunoprecipitated with anti-EGFR and anti-pY antibodies,19,20,23,25–28 including proteins associated with EGFdirected cell differentiation,25 and others affected by the EGFR inhibitors erlotinib19 and gefitinib.26 However, the overlap among

The functional annotation of phosphorylation sites remains a significant challenge in proteomics. The sorting of phosphopeptides in silico according to their phosphorylation sequence motifs,8 as a function of interactions with cognate binding partners,6,16,17,21,31 and into groups sharing temporal dynamics of abundance after EGFR activation6,8,18,21,23,24 can illuminate signaling networks. However, information on the function of individual protein phosphorylation sites remains a challenge, and strategies are needed to systematically evaluate the significance and function of phospho-proteins and phosphorylation sites. A method particularly effective for the specific enrichment of pY-containing peptides was developed by Rush et al.9 and involves the use of high-density immobilized antipY antibodies to purify pY-containing peptides from complex protein mixtures such as whole-cell extracts,9,32–38 but has not been applied to small samples containing only submicrogram amounts of protein, which is typical of an immunoprecipitate (IP).

* Author to whom correspondence should be addressed: Michael F. Moran, Hospital for Sick Children, 101 College Street, MaRS East Tower, 9-804, Toronto, ON, M5G 1L7, Canada. Tel: (416) 813-7654 x4335. Fax: (416) 813-8456. E-mail: [email protected]. † Hospital for Sick Children, and University of Toronto. ‡ McLaughlin Centre for Molecular Medicine. § Princess Margaret Hospital.

In this report, we identify protein pY sites and interacting proteins associated with the EGFR by application of a pYdirected tandem IP-mass spectrometry approach (TIPY-MS) aimed to further define the EGFR network. The approach, which is an adaptation of the Rush et al.9 method, applied to EGFR IPs, is shown to be effective in the analysis of IPs from

10.1021/pr7006363 CCC: $40.75

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Journal of Proteome Research 2008, 7, 1067–1077 1067 Published on Web 02/14/2008

research articles as few as 5 × 10 cells, including cultured cells, and xenograft tumor tissue. This revealed EGFR-associated phospho-proteins, including pY-containing proteins that were not readily identified by tandem MS analysis of EGFR IPs without pY peptide enrichment. New pY sites were uncovered in proteins known to function in the EGFR pathway including Cbl-B, Mig-6, and the Ras guanine nucleotide release factors SOS1 and SOS2. A previously hypothetical protein, the C19orf19 gene product, was shown to become tyrosine-phosphorylated at 5 sites and associated with the EGFR in response to EGF stimulation of cells. The association of C19orf19 with the EGFR was verified by co-IP of endogenous proteins in human A431 cells. These findings are presented as evidence that the TIPY-MS method, involving anti-pY peptide enrichment applied to partially purified, trypsin-digested protein complexes, is an effective approach for the concerted analysis of tyrosine phosphorylation and protein interactions, and has revealed new insights into EGFR function. 7

Experimental Procedures Constructs and Reagents. A full-length human EGFR cDNA encoding tandem C-terminal flag and green fluorescent protein (GFP) tags and a full-length human C19orf19 encoding a C-terminal myc tag were cloned into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). For IP, antiFlag-M2 beads and anti-EGFR mouse monoclonal antibody were obtained from Sigma (St. Louis, MO), anti-CDC2 mouse monoclonal was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-pY antibody reagents (anti-pTyr100, PhosphoScan Kit) for phosphopeptide purification was from Cell Signaling Technology (Danvers, MA). For Western blotting, anti-EGFR rabbit antibody and anti-pY antibody (Ab6) were obtained from Santa Cruz Biotechnology, and EMD Bios., Inc. (San Diego, CA), respectively. Toptip (200 µL) for preparing custom-made C18 tips was purchased from Canadian Life Science (Peterborough, ON). The C19orf19 rabbit polyclonal antibody was raised against a bacterially expressed, His epitope tagged C19orf19 protein, and affinity purified using purified bacterially expressed GSTC19orf19. All other chemical reagents were purchased from Sigma, and aqueous solutions were prepared using Milli-Qgrade water (Millipore, Bedford, MA). Cell Culture and Cell and Tissue Lysis. Human embryonic kidney 293 (HEK-293) cells and A431 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum. The tagged EGFR protein was stably expressed in HEK-293 cells, designated HEK-EGFR, which were cultured as described above, plus 400 µg/mL G418. For IP and Western blotting, after 10 min EGF (100 ng/mL) stimulation, cells were rinsed once with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer (LB) (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM vanadate, and a mixture of protease inhibitors). KMS12PE tumor tissue lysate was obtained by briefly homogenizing KMS12PE xenograft tumors, grown subcutaneously in nude mice, in lysis buffer, incubating at 4 °C for 30 min, and centrifuging at 15 000g at 4 °C for 10 min. The supernatants from cells and tissues were further clarified by ultracentrifugation for 60 min at 100 000g, and the resulting supernatants were used for IP. Immunoprecipitation and Western Blotting. The starting material for IP was 5–10 mg of total cellular protein in clarified lysates. This was routinely obtained from 85% confluent cells 1068

Journal of Proteome Research • Vol. 7, No. 3, 2008

Tong et al. grown in five 10-cm plates or approximately 100 mg of tumor tissue samples. These samples were subjected to IP overnight at 4 °C by using 30 µL of antiflag beads, or 5-10 µg of antiEGFR or anti-CDC2 antibodies together with 30 µL of protein G beads. Beads were washed three times with IP wash buffer (20 mM HEPES, pH 7.5, 10% glycerol, 0.1% Triton X-100, and 150 mM NaCl), and twice with HPLC-grade water. For Western blot, proteins were separated by SDS-polyacrylamide electrophoresis gels (PAGE) and transferred to an Immobilon-P membrane (Millipore). Membranes were immunoblotted using standard protocols. Tandem Immunoprecipitation Protocol. Immunoprecipitated proteins were eluted with 8 M urea, reduced with DTT, alkylated with iodoacetamide, diluted to 2 M urea, and digested with trypsin. Desalted peptides were then dissolved in 200 µL of IP buffer and pY peptides collected by incubation with 10 µL of anti-pTyr-100 beads at 4 °C overnight. The beads were washed with IP buffer, then HPLC water, and bound peptides eluted into trifluoroacetic acid (TFA). The entire eluent was analyzed by tandem MS. Peptide Desalting. Custom-made C18 chromatographic micro columns, used for desalting and concentration of peptide mixtures prior to MS analysis, were prepared with 200 µL-size Toptips containing fritted tips. After trypsin digestion, peptidecontaining solutions were acidified to a final concentration of 1% TFA and loaded into a Toptip containing approximately 30 µL of C18 beads (Honeywell Burdick & Jackson, Muskegon, MI), which was pre-equilibrated with 0.1% TFA. The micro columns were washed with 300 µL of 0.1% TFA, and then eluted by using 300 µL of 50% acetonitrile in 0.1% TFA. Eluates were dried by centrifugation under vacuum (SpeedVac). Analysis by LC-MS/MS Mass Spectrometry. Peptide mixtures from the single or tandem IP method were first separated on an automated nanoliter-scale LC system (Easy-nLC, Proxeon Biosystems A/S, Odense, Denmark), then detected using a Thermo-Fisher linear ion trap mass spectrometer system (LTQ, Thermo, San Jose, CA). Eighteen microliters of each sample was injected and automatically loaded onto a homemade C18 precolumn for concentrating and desalting, at a flow rate of 5 µL/min in HPLC buffer A (95% H2O, 5% acetonitrile (ACN) containing 0.1% formic acid). The inline analytical capillary column (75 mm × 12 cm × 6 µm tip; Proxeon) was packed with C18 resin (5 mm, 200 Å Magic C18AG, Michrom). Peptides were eluted using a linear gradient of 10–35% HPLC buffer B (5% H2O, 95% ACN containing 0.1% formic acid) over 120 min at a flow rate of 250 nL/min. The LTQ mass spectrometer was operated in a data-dependent mode consisting of a single survey MS scan followed by 6 MS/MS scans using collisionally induced dissociation (CID) as the activation mode with a collision energy of 35%. The m/z values selected for MS/MS were dynamically excluded for 30 s. The electrospray voltage applied was 2.0 kV. Both MS and MS/MS spectra were acquired using a single micro scan with a maximum fill-time of 50 and 200 ms for MS and MS/MS analysis, respectively. For MS scans, the m/z scan range was 400-1400 Thompsons. A typical phospho-peptide MS/MS spectrum is shown in Figure 3. Assigning Peptide Sequences Using SEQUEST and GPM Algorithm. Tandem mass spectra were extracted by BioWorks version 3.3. All MS/MS samples were analyzed using SEQUEST (Thermo; version 27, rev. 12) and X! Tandem (www.thegpm.org; version 2006.04.01.2). X! Tandem was set to search the ipi.HUMAN database (v3.18, 60090 entries) also assuming trypsin digestion, allowing 2 missed cleavages. SEQUEST was set to

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EGFR-Associated Phosphoprotein C19orf19

Figure 1. Expression and tyrosine phosphorylation of epidermal growth factor receptor (EGFR) in HEK-EGFR cells. (A) Anti-EGFR Western blot of three cell lines (upper panel): human tumorderived A431 (lane 1), human embryonic kidney (HEK) 293 (lane 2), and HEK-EGFR (lane 3), which is a stably transfected HEK 293 derivative that expresses approximately 2 × 106 EGFR-Flag-GFP copies per cell. Blotting with antibodies to HSP70 indicates equal protein loading in the three lanes (lower panel). (B) Anti-pY immunoblot of EGFR proteins isolated by anti-Flag immunoaffinity resin showed increased tyrosine phosphorylation of EGFR after 100 ng/mL EGF stimulation (as indicated) for 10 min (upper panel). Reprobing with anti-EGFR antibody confirmed the equivalent recovery of EGFR in both samples (lower panel).

tyrosine was specified in SEQUEST and phosphorylation of tyrosine, serine, and threonine in X! Tandem as variable modifications. Criteria for Protein Identification. Scaffold (version Scaffold-01_06_05, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm.39 Protein identifications were accepted if they could be established at greater than 90.0% probability. Protein probabilities were assigned by the Protein Prophet algorithm.40 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. The assignment of post-translational modifications (i.e., pY) was made by the search engines (described above), and verified manually. Modification by sulfation was not distinguishable from phosphorylation with the mass accuracy of these experiments. However, we have assumed these isobaric modifications to be phosphorylation as a function of the anti-pY enrichment strategy used for peptide purification.

Results

Figure 2. EGFR fusion protein peptides identified by LC-MS/MS analysis of anti-Flag single and anti-Flag/anti-pY tandem IPs. Characterized peptides are indicated by light shading, and mapped onto the complete protein sequence. Circled residues indicate pY moieties in the EGFR sequence; squares indicate phosphorylated residues within the GFP portion of the EGFR fusion protein. (A) EGFR IP sample. (B) EGFR IP + pY tandem IP. The number of unique spectra and peptides and coverage are indicated.

search the ipi.HUMAN.FLAG.fasta.hdr database (v3.18, 60090 entries) assuming the digestion enzyme as trypsin. SEQUEST and X! Tandem were searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 2.5 Da. Iodoacetamide derivative of cysteine was specified in SEQUEST and X! Tandem as a fixed modification. Phosphorylation of

Phosphotyrosine-Directed Tandem ImmunoprecipitationMass Spectrometry (TIPY-MS) Method for Protein Complex Isolation and Phosphorylation Analysis. Several methods for phospho-peptide enrichment have been developed and applied for MS sample preparation including affinity capture/chromatography,5,9,21,41–44 and chemical methods to convert phosphorylated side chains into affinity tags, protease sites, or tethers,45–51 sometimes in combinations,3 a topic recently reviewed.29,52 In the TIPY-MS method, protein complexes recovered by IP are subjected to a direct, gelfree analysis following enrichment for phosphorylated peptides by using a modified (essentially scaled-down) application of the method first described by Rush et al.9 The EGFR was recovered from the cell line HEK-EGFR, which was selected following transfection of HEK 293 cells with a flagtagged EGFR expression construct (see Experimental Procedures). These cells stably express approximately 2 × 106 copies per cell of the recombinant epitope-tagged EGFR protein, which is a level of receptor expression comparable to that found in the human tumor-derived cell line A431 (Figure 1). Western blot analysis of anti-Flag IPs showed that tyrosine phosphorylation of EGFR was dramatically increased, as expected, following EGF stimulation of HEKEGFR (Figure 1). EGFR was recovered from EGF-treated HEK-EGFR (approximately 5 × 107 cells) by IP with anti-Flag antibodies, digested with trypsin, and then analyzed by LC-MS/MS. Eighty-eight unique peptides derived from the EGFR were identified, yielding sequence coverage of 65% (Supplementary Table 1 in Supporting Information), with only a single peptide found to contain pY, observed at position Y1172 (N.B. EGFR numbering includes the 24-residue signal peptide). Figure 2 shows these peptides mapped over the sequence of the EGFR fusion protein. The poor detection of pY-containing peptides by direct analysis of the EGFR IP is not unexpected and is an indication of the low stoichiometry of EGFR tyrosine phosphorylation and the sampling nature of the MS method wherein there is a bias against low-level ions being selected for MS/ MS. Journal of Proteome Research • Vol. 7, No. 3, 2008 1069

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Figure 3. Tandem mass spectrum of the phospho-peptide GSHQISLDNPDpYQQDFFPK from EGFR identified in a tandem IP experiment. The y and b series of ions are indicated. The sequence of the peptide derived from this spectrum is shown at the top of the panel. The presence of the y7 and y8 ions provides evidence of phosphorylation on tyrosine 1172. Additional information on this peptide is contained in Table 1.

To purify pY-containing peptides, an enrichment step adapted from Rush et al.9 was used to isolate phosphopeptides contained in the trypsin-digested IP, which was estimated to contain approximately 1 pmol EGFR. LC-MS/MS analysis of the affinity-purified tryptic peptides revealed 22 unique receptor-derived peptides (Supplementary Table 2 in Supporting Information). Thirteen contained pY representing 9 distinct sites, of which 7 are known pY sites in the EGF receptor (Figure 2). Table 1 shows the 10 pY peptides derived from the EGFR that were detected by gel-free analysis of the primary IP and by the TIPY-MS (anti-Flag then anti-pY) methods. The SEQUEST Xcorr and X! Tandem -log(E-value) scores for these phospho-peptides are indicated, and all peptide and phosphosite assignments were confirmed by manual inspection of the MS/MS spectra (see Supporting Information). An MS/MS spectrum of an EGFR peptide containing residue pY 1172 is shown in Figure 3, as a representative example. Using the TIPY-MS Method To Identify pY Peptides of Endogenous Proteins from Cells and Tissue Samples. To test the feasibility of using the TIPY-MS method to study nonepitope tagged endogenous proteins, we isolated EGFR from A431 cells using a mouse monoclonal antibody that recognizes human EGFR. A431 expresses a comparable amount of EGFR protein to that of HEK-EGFR cells (Figure 1). By application of TIPY-MS, four EGFR-derived peptides were found, all of which contained pY, and located at positions 1092, 1110, 1172, 1197 (Table 1). In contrast, when the anti-EGFR IP was analyzed directly, without pY enrichment, 71 EGFR peptides were detected. In additional applications of the TIPY-MS methodology, pY-containing peptides were effectively enriched and characterized from IPs directed at the kinases Lyn and fibroblast growth factor receptor-3 (FGFR3) expressed endogenously in various human multiple myeloma-derived cell lines (data not shown). The level of EGFR protein expression (approximately twomillion copies per cell) and number of pY sites in the EGFR as described above with the HEK-EGFR and A431 cell types may be considered high relative to the average intracellular protein. Therefore, to test the feasibility of applying TIPY-MS with a 1070


Tong et al. protein target containing fewer pY sites and expressed in fewer copies than the EGFR, endogenous CDC2 kinase protein was analyzed in the human multiple myeloma tumor KMS12PE. The myeloma cells were grown as xenograft tumors in mice to further test the feasibility of using TIPY-MS for the analysis of primary tumor tissue. The CDC2 protein kinase is known to be phosphorylated at Y15, which inhibits kinase activity during the cell cycle.53 Western immunoblot analysis was used to assess the amount of CDC2 protein in cultured cells and tissue samples by comparison with recombinant GST-CDC2 standards (Figure 4), and indicated approximately 20 000 molecules of CDC2 protein per cell in KMS12PE cultured cells, and approximately 2-fold that number in the xenograft tumor tissue. Therefore, CDC2 protein was present in the cell-based samples at a moderate expression level, equivalent to approximately 100-fold fewer copies per cell than the EGFR in HEK-EGFR and A431 cells. When a KMS12PE tumor extract was analyzed by the single IP method by using anti-CDC2 antibody, 7 unique CDC2 peptides covering 31% of the entire protein were identified, and none contained pY (Figure 5 A, and Supplementary Table 3 in Supporting Information). When the TIP method was used to analyze an equivalent aliquot of the same sample, only a single tryptic peptide from CDC2 was identified (residues 10–20) and found to contain pY corresponding to the known CDC2 phosphorylation site at position 15 (pY15) (Figure 5B, and Table 1). EGFR-Associated Phospho-Proteins Identified by the TIPY-MS Method. The anti-Flag EGFR IP was found to contain several associated proteins, some of which are known to associate (directly or indirectly) with the EGFR, including Grb2, Shc, Sos1, Sos2, CBL, CBL-B, CrkL, Jak1, Sts-1, Gab1, Ack1, IRS2, and Vav2. Table 2 lists these proteins, and under the heading “single IP”, the number of unique peptides observed for each of these proteins. The values listed under the heading “tandem IP” indicate the number of unique peptides from these proteins that were observed by application of the TIP method; their pY sites are indicated in parentheses. With the exception of a single pY site found in the EGFR (pY1172; see Table 1) and one pY in CBL (pY700), none of the other proteins that co-immunoprecipitated with the EGFR were found to contain pY when analyzed in the primary IP. However, when these samples were subjected to the anti-pY enrichment step, a subset of proteins identified in the EGFR IP, including CBL, SHC, Sos1, and Sos2, were found to contain pY. The sites in SOS1 (pY1065) and SOS2 (pY1275) are to the best of our knowledge previously unreported. We and others recently published a human interactome study that included LC-MS/MS analysis of more than 1000 anti-Flag IPs from transfected HEK-293 cells under conditions similar to those described above. This study identified as “falsepositive interactors” proteins found either to frequently associate with anti-Flag beads in vector-only transfected cells (appearing in g2.5% IPs), which was a control experiment repeated 202 times, or found to frequently associate (in g5% of IPs) with a broad spectrum of Flag-bait proteins, an experiment that was repeated 832 times.15 This list of falsepositive interacting proteins provides a comprehensive control for our anti-Flag IPs that were performed in the HEK-293 cell background and using similar biochemical conditions and the same monoclonal antibody-resin conjugate. Such false-positive interactors associated with anti-Flag IPs in the current study included RBM10, hnRNP H1, RIOK1, MAP3K71P1, and DOCK4 (Table 2). HNRPH, an hnRNP complex component, was reported to interact with the protein MAP3K7IP2,54 which is

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Table 1. Phosphotyrosine-Containing Peptides Identified by LC-MS/MS Analysis Following Single and Tandem IPa peptide sequence

X! Tandem -log(e)

z

observed m/z

10.14

+2

1158.43

EGFR Peptides: Tandem IP, HEK-EGFR Cells 2. LLGAEEKEpYHAEGGKVPIK 869 3.22 3. MARDPQRpYLVIQGDER 978 2.18 4. MHLPSPTDSNFpYR 998 2.59 5. YSSDPTGALTEDSIDDTFLPVPEpYINQSVPK 1092 4.45 6. RPAGSVQNPVpYHNQPLNPAPSR 1110 3.21 7. GSHQISLDNPDpYQQDFFPK 1172 5.52 8. GSHQISLDNPDpYQQDFFPKEAK 1172 4.58 9. GSHQISLDNPDpYQQDFFPKEAKPNGIFK 1172 4.70 10. EAKPNGIFKGSTAENAEpYLR 1197 NA 11. PNGIFKGSTAENAEpYLR 1197 2.43 12. SAMPEGpYVQER 1318 2.4 13. LEYNYNSHNVyIMADKQK 1377 2.91

0.77 2.17 4.80 7.29 6.70 6.66 NA 5.08 8.77 NA 0.77 1.70

+3 +3 +3 +3 +3 +3 +3 +3 +3 +3 +2 +3

717.53 676.74 822.77 1161.02 827.66 772.58 882.80 1101.46 759.63 650.02 673.89 770.65

EGFR Peptides: Tandem IP, A431 Cells YSSDPTGALTEDSIDDTFLPVPEpYINQSVPK 1092 4.65 RPAGSVQNPVpYHNQPLNPAPSR 1110 4.67 GSHQISLDNPDpYQQDFFPK 1172 4.95 GSTAENAEpYLR 1197 4.44

2.36 6.68 5.92 5.48

+3 +3 +3 +2

1257.06 875.62 869.32 718.49

5.13

+2

633.57

1. GSHQISLDNPDpYQQDFFPK

14. 15. 16. 17.

18. IGEGTpYGVVYK

pY site

SEQUEST XCorr

EGFR Peptides: Single IP, HEK-EGFR Cells 1172 4.64

CDC2 Peptides: Tandem IP, KMS12 Tissue 15 1.68

a

Ectopically expressed EGFR protein in HEK-EGFR cells recovered by single IP (anti-Flag, row 1) and tandem IP (anti-Flag f trypsin f anti-pY, rows 2–13). Endogenous EGFR in A431 cells was analyzed by tandem IP (anti-EGFR f trypsin f anti-pY, rows 14–17). Endogenous CDC2 protein in a KMS12 xenograft tumor was analyzed by tandem IP (anti-CDC2 f trypsin f anti-pY, row 18). Peptide sequences are indicated along with the position of pY sites, the indicated peptide scores, charge state (z), and observed mass-to-charge ratios (m/z).

Figure 4. Expression and phosphorylation of the CDC2 protein kinase in cell lines and tissue samples. Upper panel: Anti-CDC2 Western blot of purified GST-CDC2 proteins (as indicated; lanes 1–3) and lysate from the human multiple myeloma plural effusion-derived line KMS12PE grown as an in vitro culture (C, lane 4) and corresponding xenograft tumor (T, lane 5). The same blot was reprobed with anti-GAPDH as a control to verify the equal loading of protein with the cell and tissue samples (lower panel).

similar to MAP3K7IP1. MAP3K7IP1 is recognized as a falsepositive, frequently found in anti-Flag IPs;15 and therefore, HNRPH is discounted as a candidate EGFR binding protein since it may represent a false-positive interaction. As an additional control, a mock IP was performed in which immobilized anti-myc antibodies were used to extract HEK-EGFR lysates and then processed and analyzed by LC-MS/MS as described above. None of the proteins associated with the antiFlag IP were identified in this control, which further indicates specificity for the EGFR and associated proteins in the Flag IP. Proteins found associated with the anti-Flag-EGFR IP and not known to frequently associate with anti-Flag IPs in HEK 293 cells15 or to our knowledge have cited connections with the EGFR include TRAP3, Peroxiredoxin-1, BCLAF1, MYCBP, CDC2, and the previously hypothetical proteins LOC144097 and

Figure 5. Peptides identified by LC-MS/MS analysis of anti-CDC2 single (A) and anti-CDC2/anti-pY tandem IP (B) experiments with a KMS12PE xenograft tumor. Characterized peptides (shaded) are shown mapped over the CDC2 sequence. The pY residue at position 15 is darkly shaded and circled. The number of unique spectra and peptides and coverage are indicated.

DKFZp761K05011. TRAP3 (Tumor necrosis factor type 2 receptor-associated protein 3, also known as TNF receptor-associated factor 2, TRAF2) is a ubiquitin ligase associated with TNF receptors and involved in modulating the NFκB pathway.55 Peroxiredoxin-1 attenuates the production of reactive oxygen species associated with ligand binding to the EGFR56 and was implicated as a tumor suppressor upregulated by the histone deacetylase inhibitor FK228 in esophageal cancer cells.57 The uncharacterized protein LOC144097 has no recognizable structural features, whereas DKFZp761K05011 is an apparent heat shock protein family member containing ATP binding and HSP90 homology regions. Confirmation of these proteins as EGFR-associated requires further validation. Eleven proteins were identified by virtue of pY peptides detected by TIPY-MS that were not detected in the analysis of the primary EGFR IP (Table 2). These proteins include known EGFR pathway components such as Mig-6 (also known as ErbB receptor feedback inhibitor 1), Gab1, Ack1, CBL-B, IRS-2, and Journal of Proteome Research • Vol. 7, No. 3, 2008 1071

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Tong et al. a

Table 2. Proteins Found To Co-IP with the EGFR

number of unique peptides protein name

EGFR CBL Grb2 SOS1 RBM10* HSP70 SHC1 TRAP3 VAV2 hnRNP H1* Peroxiredoxin-1 BCLAF1 LOC144097 DOCK4* MYCBP DKFZp761K0511 CRKL MAP3K7IP1* RIO kinase 1* JAK1 pro-EGF HNRPH* Sts-1 SOS2 Mig-6 GAB1 ACK1 CDC2 c-FER CBL-B FLJ20035 C19orf19 HCFC1 IRS-2 RBM41

accession numbers single IP

IPI00018274 IPI00027269 IPI00021327 IPI00020131 IPI000375731 IPI00304925 IPI00021326 IPI00513796 IPI00104050 IPI00004977 IPI00013881 IPI00000874 IPI00006079 IPI00106955 IPI00006024 IPI00554793 IPI00334775 IPI00004839 IPI00019459 IPI00171257 IPI00011633 IPI00000073 IPI00026230 IPI00154910 IPI00020134 IPI00004399 IPI00031068 IPI00442025 IPI00026689 IPI00029263 IPI00292856 IPI00217606 IPI00166190 IPI00019848 IPI00464978 IPI00306343

88 17 15 12 11 9 8 7 7 6 6 4 4 3 3 3 3 3 3 3 2 2 2 1 0 0 0 0 0 0 0 0 0 0 0

tandem IP

22 (13 with pY) 2 (pY674 PY700) 0 1 (pY1065) 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 (pY1275) 2 (pY394, pY458) 2 (pY373, pY659) 2 (pY937 or pY938) 1 (pY15) 1 (pY229) 1 (pY889) 1 (pY793, pY796) 1 (pY207) 1 (pY1923) 1 (pY825) 1 (pY302)

a Shown are the number of unique peptides for the indicated proteins found by LC-MS/MS analysis of anti-Flag IPs (Single IP, column 3), and by using the TIPY-MS method (Tandem IP, column 4). pY positions are indicated in parentheses, except for EGFR. For EGFR, 13 of the 22 unique peptides contained pY. Known EGFR binding proteins are in boldface, and proteins considered false-positives (as explained in the text) are denoted with asterisks. Column 2 provides the International Protein Index accession numbers (www.ebi.ac.uk/IPI). The last 11 proteins on the list were identified based soley on pY-containing peptides detected by the TIPY-MS method and were not detectable in single IPs.

proteins not previously described as EGFR associated including CDC2, c-Fer, HCFC1, RBM41, and the (until now) hypothetical proteins C19orf19 and FLJ20035 (see Table 2). The identification of these phosphopeptides illustrates that tyrosine-phosphorylated peptides derived from the primary IP, but present in amounts too low to be selected for analysis by the tandem MS sampling algorithm, are effectively enriched by the anti-pY step. These modifications appear to be EGF-dependent since no pYcontaining peptides were detected when the TIP method was used to analyze Flag-EGFR IPs from HEK-EGFR cells not stimulated with EGF (data not shown). Among the EGF-induced group of phospho-peptides detected only by tandem IP (Table 2), seven pY sites are previously unreported: Mig-6 pY458, c-Fer pY229, CBL-B pY889, FLJ20035 pY793, and pY796 (i.e., two sites on one peptide), C19orf19 pY207, HCFC1 pY1923, and RBM41 pY302. The observation of the CDC2 pY15 peptide is coincidental to our examination of it described above, and not due 1072

Table 3. Information Related to Proteins Identified by LC-MS/ MS Analysis of EGFR Single and Tandem IPa


protein name

accession no.

length (AA)

Single IP IPI00018274 1464 IPI00027269 906 IPI00021327 217 IPI00020131 1333 IPI00304925 641 IPI00021326 SHC1 IPI00513796 584 TRAP3 IPI00104050 955 vav 2 IPI00004977 839 Peroxiredoxin-1 IPI00000874 199 BCLAF1 IPI00006079 920 LOC144097 IPI00106955 381 MYCBP IPI00554793 102 DKFZp761K0511 IPI00334775 737 CRKL IPI00004839 303 JAK1 IPI00011633 1156 EGF precursor IPI00000073 1207 Sts-1 IPI00154910 649 EGFR CBL Grb2 SOS1 HSP70

EGFR CBL

Tandem IP IPI00018274 1464 IPI00027269 905

coverage (%)

no. unique peptide

no. unique spectra

65 29 64 15 27

88 17 15 12 9

192 19 26 13 9

33 12 14 28 8 22 50 7 21 5 3 3

8 7 7 6 4 4 3 3 3 3 2 2

10 9 7 6 4 4 3 3 4 3 2 2

18 5

22 2

48 4

a Protein identification probabilities were 100% according to ProteinProphet.40

to carryover between samples destined for MS analysis. Identified proteins containing pY but not previously implicated in EGFR signaling include FLJ20035, which has a helicase homology domain and DEAD box found in helicases, HCFC1, which is a transcriptional regulator, and RBM41, which is an uncharacterized protein with a RNA binding motif. While these proteins are not reported to frequently interact with anti-Flag IPs from HEK-293 cells,15 they remain unvalidated as EGFR network/pathway components based on our MS-based studies alone. Additional information pertaining to protein identifications, and peptides analyzed by MS/MS and described above, is listed in Tables 3 and 4. The C19orf19 Protein Is a Novel Phospho-Protein in the EGFR Pathway. To support the contention that the TIPY-MS approach enables the identification of functionally related proteins, we sought to validate the apparent connectivity between the EGFR and C19orf19 (UniProtKB/Swiss-Prot entry Q3SX64), an uncharacterized protein previously identified as hypothetical. When the amount of starting material (HEK-EGFR cells) was increased 4-fold over that used in the experiments described above, a single, nonphosphorylated C19orf19-derived peptide was detected in the trypsin-digested anti-Flag-EGFR IP (Table 5). While not quantitative, this would be consistent with C19orf19 being only a minor component of the anti-EGFR immune complex. Sequence analysis of C19orf19 indicates that, relative to the human proteome, it is proline-rich; 48 (16.6%) of its 289 residues are prolines. Kyte and Doolittle analysis58 for hydropathy indicates that C19orf19 is a hydrophilic protein. It contains three 27-residue DUF1309 sequence motifs, which are uncharacterized, but recognized in several eukaryotic proteins of unknown function. To further investigate the interactions and phosphorylation of C19orf19, expression constructs and antibodies were pre-

research articles

EGFR-Associated Phosphoprotein C19orf19 a

Table 4. Information Related to Single Peptides and Phospho-Peptides protein name

accession numbers

sequence

position

SEQUEST Xcorr Score

X! Tandem -log(e)

z

m/z

SOS2 SOS2 SHC1 SOS1 CBL CBL Mig-6 Mig-6 GAB1 ACK1 ACK1 CDC2 c-FER CBL-B FLJ20035 C19orf19 HCFC1 IRS-2 RBM41

IPI00020134 IPI00020134 IPI00513796 IPI00020131 IPI00027269 IPI00027269 IPI00004399 IPI00004399 IPI00031068 IPI00442025 IPI00442025 IPI00026689 IPI00029263 IPI00292856 IPI00217606 IPI00166190 IPI00019848 IPI00464978 IPI00306343

SAEEKNNWMAALISLHYR RCpY1275VLSSSQNNLAHPPAPPVPPR KQMPPPPPCPGRELFDDPSpY99VNVQNLDK KISpY1065SRIPESETESTASAPNSPR LPPGEQcEGEEDTEpY700MTPSSRPLRPLDTSQSSR IKPSSSANAIpY674SLAAR KHLSpY459VVSP VSSTHpY394YLLPERPPYLDKYEK SSGSGSSVADERVDpY659VVVDQQK KVSSTHpY937YLLPERPSYLER VSSTHYpY938LLPERPSYLER IEKIGEGTpY15GVVYK ALKGIFDEpY229SQITSLVTEEIVNVHK TSQDpY889DQLPScSDGSQAPARPPKPR ELLDVVDKNESAVIVAPTSSGKTpY793ASpY796YCMEK TPPARPPQDPAEIPGPGQpY207DSPDANTYR IIEpY1923SVYLAIQSSQAGGELKSSTPAQLAFMR SYKAPYTCGGDSDQpY825VLMSSPVGR IPMFSSpY302NPGEPNKVLYLK

528 1273 80 1062 656 664 455 389 645 931 932 7 221 885 770 189 1920 811 296

4.1 2.15 4.12 2.63 2.4 4.08 2.32 2.59 2.54 2.85 2.88 2.64 2.21 2.1 2.81 4.38 2.01 3.73 2.3

4.75 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

2 3 3 3 3 3 2 3 3 3 3 2 3 3 3 3 3 3 3

1067.57 879.87 1107.22 863.45 1277.63 577.41 555.23 893.99 798.36 807.38 764.66 818.28 971.8 947.09 1243.67 1029.97 1147.32 907.21 759.98

a Peptide identification probabilities were more than 95% as determined by PeptideProphet.39 Numbers within peptide sequences are included adjacent to pY residues to indicate their position.

Table 5. Information Associated with Single Peptides or Phospho-Peptides Associated with the C19orf19 Proteina C19orf19 sequence

SEQUEST Xcorr

X! Tandem -log(e)

z

m/z

115SRGPEVTPGPGAYSPEK 33KSCGTATLENGSGPGLpY49VLPSTVGFINHDCTR 65VASPApY70SLVR 75RPSEAPPQDTSPGPIpY90FLDPK 189TPPARPPQDPAEIPGPGQpY207DSPDANTYR 189TPPARPPQDPAEIPGPGQYDSPDANTpY215RQR

5.67 5.02 2.13 NA 4.63 3.29

6.32 8.92 1.13 6.36 5.60 NA

2 3 2 3 3 3

865.66 1163.81 572.10 799.02 1030.63 1124.34

a Numbers before peptide sequences in column 1 indicate the position in the protein of the first residue of the peptide. Numbers within peptide sequences are included adjacent to pY residues to indicate their position. The nonphosphorylated peptide that begins at residue 115 was detected by analysis of an anti-Flag-EGFR IP. The five pY- containing peptides are as indicated in Figure 8. Peptide identification probabilities were more than 95% as determined by PeptideProphet.39

pared. Affinity-purified anti-C19orf19 antibodies purified from rabbit serum following the fourth boost with antigen were used to probe human cell lines and murine tissues. Brain, kidney, muscle, and lung were selected for analysis based on predictions of C19orf19 expression by one or more of expression array, electronic Northern, and SAGE, as compiled by Gene Card59 (http://www.genecards.org/cgi-bin/carddisp.pl?gene) C19orf19). Expression was not predicted for lung tissue, which was therefore included as a possible negative control. Myctagged C19orf19 protein, present in transiently transfected HEK 293 cells, was detected by immunoblotting with the affinitypurified rabbit anti-C19orf19 antibodies, and thereby served as a positive control (Figure 6A, lane 1). A band migrating near myc-tagged protein was observed in both HEK 293 cells (Figure 6A, lane 2) and the human epidermoid carcinoma line A431 (lane 3), which is likely endogenous C19orf19 protein. Endogenous C19orf19 protein was readily identified in mouse kidney (Figure 6A, lane 5), while a more diffuse co-migrating band was observed in the muscle sample. A very faint signal was detected in brain tissue, and no signal was detected in lung extract. In addition to these cells and tissues, we have detected by Western blotting evidence for C19orf19 expression in various cell lines including murine C2C12 myoblasts, Rat1 fibroblasts, human myelomas KMS11 and KMS12, human neuroblastoma NBE2, human lung cancer A549, human colon cancers HTC116 and HTC15, human breast cancers T470 and 5637, but not normal human fibroblasts, Rat

PC12, and human pancreatic cancer BxPC3 or HPAC cells. More comprehensive analyses will be needed to fully characterize C19orf19 expression. We conclude that endogenous C19orf19 is expressed in kidney, which is consistent with its identification in the EGFR IP from the kidney-derived HEK-EGFR cells as well as in Madin-Darby canine kidney (MDCK) cells (not shown), and supports our choice to further characterize C19orf19 in the HEK 293 and monkey kidney-derived COS-7 cells described below. To further validate the C19orf19-EGFR interaction, we sought to test for the reciprocal co-IP of ectopic C19orf19, C-terminally tagged with a myc epitope, with the EGFR in HEK and COS cells. As shown in Figure 6B, myc-tagged C19orf19 was efficiently detected by anti-myc blotting of HEK 293 cells transiently transfected with the C19orf19myc construct (Figure 6B, lane 1). When these cells, which do not express detectable levels of EGFR, were subjected to IP with anti-EGFR antibodies, C19orf19myc protein was not detected by Western blotting (Figure 6B, lane 2), indicating an absence of nonspecific interaction of C19orf19 with the anti-EGFR reagent. However, when transiently expressed in HEK-EGFR cells, ectopic C19orf19myc was efficiently recovered by anti-EGFR IP, but only when the cells were stimulated with EGF (compare lanes 3 and 4,Figure 6B). This demonstrates that the association of C19orf19 with the EGFR is a consequence of EGFR activation. A reciprocal experiment is shown in Figure 6C. EGFR was found to co-IP with C19orf19myc in EGF-stimulated, but not unstimulated, HEK-EGFR cells. Further Journal of Proteome Research • Vol. 7, No. 3, 2008 1073

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Tong et al.

Figure 6. C19orf19 expression, association with EGFR, and EGF-induced tyrosine phosphorylation. (A) Affinity-purified rabbit antibodies directed against recombinant C19orf19 were used to probe the indicated cell lines (lanes 1–7) and tissues for expression of C19orf19 as indicated. Lane 1 contains a cell lysate from transiently transfected HEK 293 cells expressing C19orf19myc protein, indicated by the arrow. (B-D) HEK 293 cells and HEK-EGFR cells untransfected and transiently transfected with myc-tagged C19orf19 (C19orf19myc, as indicated) and treated without (-) or with (+) EGF for 10 min were immunoprecipitated with anti-EGFR or anti-Myc antibody as indicated, and immunoblotted with the indicated antibodies; samples of cell lysate (L, as indicated) not subjected to IP were run as controls. (B) Co-IP of C19orf19 with EGFR in an anti-EGFR IP from cells treated with EGF. (C) Co-IP (anti-myc) of EGFR and C19orf19myc with EGFstimulated HEK-EGFR, but not HEK 293 cells that lack EGFR expression. (D) Anti-pY Western blot indicates EGF-induced tyrosine phosphorylation of C19orf19myc in HEK-EGFR but not HEK 293 cells. (E) Anti-myc Western blot indicates expression of C19orf19myc in transiently transfected Simian COS-7 cells (lanes 1 and 2), and anti-pY Western blot indicates tyrosine phosphorylation of C19orf19myc following EGF-stimulation of transfected COS-7 cell, which express endogenous EGFR at a level ∼15% of A431 (lanes 3 and 4). (F) Western blot with EGFR antibodies (upper panel) and C19orf19 polyclonal antibodies (lower panel). Control IP with nonspecific immunoglobulin (Ig, lane 1) and IP with affinity-purified C19orf19 polyclonal antibodies (lanes 2 and 3) indicate the recovery of endogenous EGFR together with endogenous C19orf19 from EGF-stimulated A431 cells (lane 3), and to a lesser extent from unstimulated cells (lane 2).

analysis of these immune complexes by anti-pY Western blotting indicated that tyrosine phosphorylation of C19orf19myc was induced by EGF treatment (Figure 6D). Tyrosine phosphorylation of myc-tagged C19orf19 was observed in transiently transfected COS-7 cells (Figure 6E). The EGF-induced tyrosine phosphorylation of C19orf19myc in COS-7 cells was less robust than in the HEK-EGFR system likely due to the much lower level of expression of endogenous EGFR in the COS-7 cells. As a more rigorous examination of the EGFR-C19orf19 interaction, and to eliminate any possibility their observed interaction was a function of epitope tags or overexpression, we tested for the association of endogenous C19orf19 and EGFR in human epithelial carcinoma A431 cells. C19orf19 protein was isolated by IP with affinity purified polyclonal antibodies to C19orf19, as was a detectable level of EGFR (Figure 6F, lane 2). Stimulation of A431 cells with EGF did not affect the recovery of C19orf19 in the anti-C19orf19 IP, but induced a pronounced increase in the recovery of EGFR (Figure 6F, lane 3). These findings support our conclusion that C19orf19 interacts directly or indirectly (i.e., possibly bridged through another molecule) with the EGFR, and that the co-IP of C19orf19 with ectopically expressed EGFR (described above) is not a function of the flag or GFP sequences in the receptor fusion protein. TIPY-MS Analysis Indicates EGF-Induced Phosphorylation of C19orf19 at Five Tyrosine Sites. The TIPY-MS method was applied to provide additional information into the effect of EGF stimulation on C19orf19 phosphorylation, and its association with the EGFR. C19orf19 was isolated from transiently transfected HEK-EGFR cells using myc-specific antibodies and analyzed by LC-MS/MS. Twenty-four unique 1074


Figure 7. Peptides derived from C19orf19 identified by LC-MS/ MS analysis of anti-myc single IP (A) and anti-myc/anti-pY tandem IP (B) from C19orf19myc-transfected HEK-EGFR cells. The number of unique spectra and peptides and coverage are indicated. Peptides identified by MS/MS are indicated by shading and mapped onto the C19orf19 sequence. C19orf19 residues modified by phosphorylation on tyrosine (5 sites) are shaded dark and circled, and were found only following the double IP protocol (B).

peptides representing 86% of the total protein were detected by this gel-free analysis, and no pY-containing peptides were observed (Figure 7A). When the trypsin-digested primary IP was further purified using anti-pY, 14 unique C19orf19 peptides were observed, and this set included 5 distinct sites of tyrosine


Figure 8. EGF-induced phosphorylation of C19orf19. Representative spectra demonstrating tyrosine phosphorylation of C19orf19 at positions Y70 (top three panels) and Y90 (bottom panel). The two middle panels show expanded regions of the indicated spectrum for C19orf19 phosphopeptide VASPApYSLVR to better reveal the definitive y and b ions. Additional information is listed in Table 5.

phosphorylation (Figure 7B, and Supporting Information), including pY207, which was also observed in the TIPY-MS analysis of the EGFR (see Table 2). Also detected among the pY peptides derived from the C19orf19 IP were two EGFR phospho-peptides containing pY1172 and pY1197, which reflects the association of C19orf19 with the activated EGFR. Table 5 provides additional information on the single C19orf19 peptide found to co-IP with the EGFR, and the C19orf19 phosphopeptides identified by the anti-C19orf19/anti-pY TIP analysis. Figure 8 shows representative spectra indicating phosphorylation of C19orf19 at postions Y70 and Y90.

Discussion Signaling by growth factor receptors including the EGFR is, to a great extent, a function of coordinated protein–protein interactions and phosphorylation. MS is an efficient tool to define the components of protein complexes and to identify protein phosphorylation sites. The tandem IP method described in this study, in which MS/MS analysis was used to characterize

research articles peptides isolated by anti-pY extraction of protease-digested EGFR IPs, was an effective approach to analyze both protein– protein interactions and phosphorylations associated with EGFR signaling. There is the possibility of a functional connection with the EGFR tyrosine kinase, when associated proteins are themselves phosphorylated on tyrosine. This provides a rationale to further test for functional relationships. This was the case with the C19orf19 protein, in which functional connections with the EGFR were demonstrated in three respects beyond its original anti-Flag-EGFR IP association. First, reciprocal IPs in which C19orf19 was the epitope-tagged bait protein contained the EGFR. Furthermore, the interaction was verified by using anti-C19orf19 antibodies to isolate the complex containing endogenous EGFR and C19orf19 in A431 cells. Second, the association of C19orf19 with the receptor was induced by EGF treatment of cells. Third, EGF stimulation of cells caused C19orf19 to become tyrosine-phosphorylated at 5 distinct tyrosine residues (Figure 7, Table 5), and with kinetics similar to EGFR phosphorylation at Y1068 and ERK phosphorylation (not shown). These findings indicate C19orf19 is part of the EGFR network and suggest it may be subject to regulation by tyrosine phosphorylation. C19orf19 is predicted to contain 289 amino acids and have a molecular mass of 31 kDa. It contains three 27-residue DUF1309 motifs, which have no defined structure or function. A more comprehensive characterization of C19orf19 is an objective of our ongoing investigations. The anti-pY precipitation of phospho peptides, adapted from Rush et al.,9 provided an important purification step required to increase the relative concentration of the modified peptides such that they are more likely to be selected for MS/MS in the ion trapping instrument. This facilitated the identification of associated proteins present in low stoichiometric amounts. Such minor, associated proteins may reflect specific functional complexes. For example, among the set of EGFR pathway components that were only detected by the TIPY-MS method are Mig-6, Ack1, and CBL-B, each of which is implicated in the internalization or down-regulation of the EGFR, which likely involves only a subset of EGF receptors in the cell and captured by the IP. The TIP approach may be particularly applicable with MS platforms such as the LTQ ion trap running in a datadependent mode wherein minor abundance ions may not be selected for MS/MS analysis. We detected 7 pY sites in the EGFR by TIP analysis in conjunction with the LTQ ion trap. Wu et al.23 detected 7 pY sites in the EGFR without a phosphopeptide enrichment step using a higher resolution instrument (LTQ-Orbitrap). Only four are common to both sets: Y1092, Y1110, Y1172, and Y1197. Sites found by Wu et al.23 and not detected by the TIPY-MS approach are Y1016, Y1069, and Y1138, while the TIPY-MS method found Y869, Y978, and Y998, which were not observed by Wu et al.23 Therefore, a total of 10 unique EGFR pY sites, all previously described in the literature, are described in the union of the two data sets. The fact that these two data sets are not completely overlapping illustrates the benefit of complementary approaches, which, in this instance, include different proteases, EGFR antibodies, MS instrumentation, and EGFR source material. We conclude that TIPY-MS, including its application in an iterative manner as was done to analyze C19orf19 following its initial discovery in an EGFR IP, may serve as an effective and generally applicable approach to define functional connections involving protein interactions and phosphorylation. Journal of Proteome Research • Vol. 7, No. 3, 2008 1075

research articles Acknowledgment. We thank Sarah Wang and Jieran Li for technical support; Thomas Kislinger and Brian Raught for helpful comments on the manuscript; Warren Shih, Shawna Organ, and Drs. Ming Tsao, David Kaplan, Dan Lin, Parveen Shama, Victor Mao, James Pan, Tak Mak, and David Hedley for cell lines. M.F.M. is supported by The Canada Research Chairs Program, The Hospital For Sick Children Foundation, The Canadian Institutes for Health Research, Canadian Cancer Society, and National Cancer Institute of Canada. Supporting Information Available: Supplementary Table 1, EGFR peptides identified by single immunoprecipitation from HEK-EGFR cells; Supplementary Table 2, EGFR peptides identified by tandem immunoprecipitation from HEKEGFR cells; Supplementary Table 3, CDC2 peptides identified by single IP approach; Supplementary MS/MS spectra, spectra for all phospho-peptides and peptides associated with singlepeptide-based protein identification. Spectra are labeled to indicate the figure or table with which they are associated, and are presented in the same order as they appear in these figures/ tables. MS/MS searches and phosphosite assignment are described in Experimental Procedures. In some instances, spectra are presented in more than one image to zoom into regions of low intensity. This material is available free of charge via the Internet at http://pubs.acs.org.

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