Table 3. Peptides from the Newly Identiﬁed Src Substrates That Were Phosphorylated by c-Src on Peptide Microarrays accession no. protein phosphopeptide 1 NP_003893 FUSE binding protein 1 2 NP_003676 FUSE binding protein 2 1. EVRNEYGSRIG 2. RQQAAYYAQTS 1. GDRNEYGSRIG 2. AYYSHYYQQPP 3. RQQAAYYGQTP BCL2-associated transcription 1. YHRGGYRPVWN factor 1 2. EETEDYRQFRK 3. GRGRGYYQGGG 4. NGSSRYSPSQN 5. RGRGYYQGGGG 6. RSSSPYSKSPV RNA binding motif protein 10 1. ARGSSYGVTST 2. EPPVDYSYYQQ 3. DRTGRYGATDR TRK-fused Gene 1. NEDITYDELVL 2. QMYQQYQQQAG 1. NDIFRYQDEVF RasGAP SH3-domain-binding protein Ataxin 2 related protein A-Kinase anchor protein 8 Thyroid hormone receptor associated protein 3 1. GQQGKYRGAKG 1. RPSYSYDYEFD 1. NYRQAYSPRRG 2. NHPRVYQNRDF 3. SGGAAYTKRYL 4. GGYGNYRSNWQ 5. GTPAGYGRGRE 1. AGRGNYDAFRG research articles Amanchy et al. Figure 5. Experimental validation of tyrosine phosphorylation of proteins obtained from c-Src kinase overexpression in 293T cells. (A) In vitro kinase assays using GST tagged proteins and c-Src using a rabbit reticulocyte in vitro transcription and translation system. (B) 293T cells were cotransfected with genes of interest along with either empty vector PCMVtag4A or with c-Src. Culture media was changed 12 h after transfection and cells were serum- starved for 12 h and lysed 48 h after transfection. Proteins were immunoprecipitated using anti-Flag antibodies and Western blotting was performed using phosphotyrosine antibodies and reprobed. (C) Validation of a subset of proteins in PDGF signaling. NIH3T3 cells have been grown to conﬂuence and serum-starved for 12 h followed by stimulation with PDGF-BB (100 ng/mL for 5 min) and PDGF stimulation after treatment with SU6656 (2 µ M for 1 h prior to lysis or stimulation), and cell lysates were subjected to immunoprecipitation using anti-phosphotyrosine antibodies, probed with respective antibodies, and reprobed in whole cell lysates. (Figure 6A). Intensity values were obtained using GenePix software as described under Experimental Procedures. We normalized the intensity values and compared the log 2 intensities of the WT peptides against their corresponding MUT peptides. The intensity values from 3 different experiments were averaged individually for WT and MUT peptides and plotted (Figure 5B). The spots in the upper right quadrant were taken as true positives (Figure 6B). The false positive rates (FPR) were calculated by assuming that those peptides for which MUT intensity exceeded WT intensity 3908 Journal of Proteome Research Vol. 7, No. 9, 2008 3 NP_055554 4 NP_005667 5 NP_006061 6 NP_005745 7 NP_663760 8 NP_005849 9 NP_005110 10 NP_877952 Arsenate resistance protein ARS2 2. QGLMPYGQPRP 3. HSSDPYHSGYE 4. KRYNDYKLDFR 11 NP_006833 Splicing factor 3B subunit 2 1. HGDLYYEGKEF 2. EEPEIYEPNFI 3. QREESYSRMGY 12 NP_055983 hypothetical protein LOC23211 1. GRGRGYRGRGS 2. DRYNSYNRPRP 3. RKYREYSPPYA were not phosphorylated, and that the variation of WT-MUT intensities for those points was representative of nonphosphory- lated peptides (Figure 6C). On the basis of the calculated FPRs, a line was drawn which separates the true positives from the remainder of the peptides. From this analysis, we have identiﬁed phosphorylation sites on 12 out of 14 proteins that were spotted on peptide microarrays. Peptides containing multiple tyrosines were mutated systemati- cally and all the tyrosine containing peptides were looked at along with their corresponding mutated counterparts to deduce the correct phosphorylated peptide. A total of 34 peptides from 12 proteins (Table 3) out of 312 peptides from 14 proteins spotted (Supplementary Table 2) were phosphorylated by c-Src in our analysis. This included 6 phosphopeptides from Bcl2-associated transcription factor, 5 from Thyroid hormone receptor associated protein 3, and 4 phosphopeptides from ARS2. We did not detect phosphorylation on any of the peptides derived from two proteins, Zinc ﬁnger, CCHC domain containing 8 and Splicing factor proline/glutamine-rich. Thus, peptide arrays allowed assignment of phosphorylation sites in a high-throughput fashion and also served as an additional validation step for potential substrates identiﬁed from our SILAC experiments. Because of the tremen- dous potential and promise that peptide microarrays hold for the identiﬁcation kinase-substrate identiﬁcation, studies are ongoing to spot the peptides from other proteins identiﬁed in this and other mass spectrometry based studies to identify the kinase- speciﬁc phosphorylation sites and build phosphorylation motifs. Conclusions This study describes identiﬁcation of substrates of a nonre- ceptor tyrosine kinase using a combination of proteomic