Identiﬁcation of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays Ramars Amanchy,† Jun Zhong,† Henrik Molina,†,‡ Raghothama Chaerkady,†,# Akiko Iwahori,† Dario Eluan Kalume,†,⊥,§ Mads Grønborg,† Jos Joore,§ Leslie Cope,| and Akhilesh Pandey*,† McKusick-Nathans Institute of Genetic Medicine and the Departments of Biological Chemistry, Pathology and Oncology, Johns Hopkins University, Baltimore, Maryland 21205, Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Center for Experimental Bioinformatics, Odense 5230, Denmark, Pepscan Systems, Edelhertweg 15, 8219 PH Lelystad, The Netherlands, and Sidney Kimmel Comprehensive Cancer Center and the Department of Biostatistics, Bloomberg School of Public Health, and Johns Hopkins University, Baltimore, Maryland 21205 Received March 17, 2008 c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human embryonic kidney 293T cells. We have identiﬁed 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c-Src using in vitro kinase assays and cotransfection experiments. Signiﬁcantly, using a c-Src speciﬁc inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues that are phosphorylated by c-Src on the novel c-Src substrates, we designed custom peptide microarrays containing all possible tyrosine-containing peptides (312 unique peptides) and their mutant counterparts containing a Tyr f Phe substitution from 14 of the identiﬁed substrates. Using this platform, we identiﬁed 34 peptides that are phosphorylated by c-Src. We have demonstrated that SILAC-based quantitative proteomics approach is suitable for identiﬁcation of substrates of nonreceptor tyrosine kinases and can be coupled with peptide microarrays for high-throughput identiﬁcation of substrate phosphopeptides. Keywords: Phosphorylation • SILAC • PDGF • Quantitative mass spectrometry • Systems biology Introduction Most signaling pathways include protein kinases and their substrates that serve as means to amplify signals from extra- cellular signals and other stimuli. However, the precise con- nectivity between protein kinases and their downstream sub- strates has not been fully elucidated for most protein kinases. c-Src is a classic nonreceptor tyrosine kinase that has been implicated in regulation of cytoskeletal rearrangement and cell adhesion networks that control cell migration, cell proliferation * Corresponding author: Tel, 410-502-6662; fax, 410-502-7544; e-mail, pandey@jhmi.edu. † McKusick-Nathans Institute of Genetic Medicine and the Departments of Biological Chemistry, Pathology and Oncology, Johns Hopkins University. ‡ University of Southern Denmark. ⊥ Current address: Department of Tropical Medicine, Oswaldo Cruz Foundation - FIOCRUZ, Rio de Janeiro, RJ, 21040-900, Brazil. § Pepscan Systems. | Sidney Kimmel Comprehensive Cancer Center and the Department of Biostatistics, Bloomberg School of Public Health, and Johns Hopkins University. # Institute of Bioinformatics, International Technology Park. 3900 Journal of Proteome Research 2008, 7, 3900–3910 Published on Web 08/13/2008 and cell survival.1 One vital step in understanding the role of c-Src kinase in cellular transformation and signaling is sys- tematic identiﬁcation of all of its potential cellular substrates involved in these processes. Recent studies based on advances in mass spectrometry- based proteomics have provided large-scale catalogs of phos- phorylation sites.2–5 However, determination of kinases re- sponsible for these phosphorylation events is not an easy task owing to the transient interaction between kinases and their substrates. Chemical and genetic approaches have been previ- ously used to identify c-Src substrates. Such studies include use of an ATP analogue that is a speciﬁc substrate for an analogue-speciﬁc allele of v-Src,6 screening of cDNA expression libraries with anti-phosphotyrosine antibodies7 and use of mutant inducible forms of c-Src.8 To date, several c-Src substrates as well as interactors have been reported. Human Protein Reference Database (HPRD)9 provides a list of 132 c-Src mediated phosphorylation sites in 64 known substrates along with 204 proteins that interact with c-Src. 10.1021/pr800198w CCC: $40.75  2008 American Chemical Society