research articles Amanchy et al. Figure 3. MS spectra of 6 proteins identiﬁed as Src substrates by SILAC. The 3 spectral peaks in each ﬁgure represent the mass shift of the same peptide. The relative increase in intensity ratios between light to heavy are represented below in parentheses. (A) A doubly charged peptide from cortactin (1:7); (B) a triply charged peptide from NICE-4 (1:7); (C) a doubly charged peptide from Bcl2- associated transcription factor (1:7.5); (D) a doubly charged peptide from FUSE-binding protein 1 (1:8); (E) a doubly charged peptide from FUSE-binding protein 2 (1:4); (F) a doubly charged peptide from RNA binding motif 10 (1:7.5). apoptotic transcriptional repressor43 localized to the nucleus, whose function is still under investigation. RNA binding motif 10 (RBM10) is an RNA binding protein with a zinc ﬁnger domain, associated with the expression of Bax family members in breast cancers and VEGF.44 This was the ﬁrst time BTF and RBM10 were identiﬁed as a tyrosine-phosphorylated proteins and the ﬁnding that they are components of PDGF signaling downstream of c-Src might help to understand the role of BTF and RBM10 in growth factor receptor signaling. Although we have observed tyrosine phosphorylation of a subset of proteins in PDGF signaling, it is not possible to validate all candidates in any proteomics experiment because of the following reasons: (i) Availability of good immunoprecipitating antibodies against these proteins is limited, and hence, we have tagged a subset 3906 Journal of Proteome Research Vol. 7, No. 9, 2008 of these proteins to show that these are indeed substrates; and (ii) To validate these proteins in a speciﬁc signaling pathway, it is not easy to predict in which signaling pathway(s) these proteins are involved downstream of c-Src. However, studies are currently in progress on a subset of proteins to show their physiological relevance and also to identify tyrosine-phospho- rylated proteins in growth factor signaling pathways down- stream of Src kinase. Development of Peptide Microarrays for High-Throughput Validation of c-Src Substrates. Although we have established above that a number of novel proteins are potential substrates of c-Src, the exact residues that undergo phosphorylation have not been identiﬁed in most of these instances. Hence, we developed a custom peptide microarray as a platform to rapidly