Ramars Amanchy

Human protein reference database as a discovery resource for proteomics

The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein-protein interactions, post-translational modifications, enzyme-substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease.

Global Impact of Oncogenic Src on a Phosphotyrosine Proteome

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.

Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications.

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of a tag into proteins for relative quantitation by mass spectrometry. SILAC is a simple, yet powerful, method for investigating the dynamics of protein abundance and posttranslational modifications. Here, we provide detailed instructions for using this method to study protein complexes, protein-protein interactions, and the dynamics of protein abundance and posttranslational modifications. We expect that SILAC will become a routine technique because of its applicability to most areas of cell biology. We have also developed a Web site (http://www.silac.org) to provide researchers with updated information about this method and related resources.

Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

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 → 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.

Identification of c-Src tyrosine kinase substrates in platelet-derived growth factor receptor signaling

c‐Src non‐receptor tyrosine kinase is an important component of the platelet‐derived growth factor (PDGF) receptor signaling pathway. c‐Src has been shown to mediate the mitogenic response to PDGF in fibroblasts. However, the exact components of PDGF receptor signaling pathway mediated by c‐Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Src‐family kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c‐Src kinase substrates in PDGF receptor signaling. This included 23 known c‐Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of 20 novel Src‐family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining 15 proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src‐family kinase substrates – Calpain 2, Eps15 and Trim28 – in a c‐Src‐dependent fashion.

Identification of tyrosine-phosphorylation sites in the nuclear membrane protein emerin

Although several proteins undergo tyrosine phosphorylation at the nuclear envelope, we achieved, for the first time, the identification of tyrosine‐phosphorylation sites of a nuclear‐membrane protein, emerin, by applying two mass spectrometry‐based techniques. With a multiprotease approach combined with highly specific phosphopeptide enrichment and nano liquid chromatography tandem mass spectrometry analysis, we identified three tyrosine‐phosphorylation sites, Y‐75, Y‐95, and Y‐106, in mouse emerin. Stable isotope labeling with amino acids in cell culture revealed phosphotyrosines at Y‐59, Y‐74, Y‐86, Y‐161, and Y‐167 of human emerin. The phosphorylation sites Y‐74/Y‐75 (human/mouse emerin), Y‐85/Y‐86, Y‐94/Y‐95, and Y‐105/Y‐106 are located in regions previously shown to be critical for interactions of emerin with lamin A, actin or the transcriptional regulators GCL and Btf, while the residues Y‐161 and Y‐167 are in a region linked to binding lamin‐A or actin. Tyrosine Y‐94/Y‐95 is located adjacent to a five‐residue motif in human emerin, whose deletion has been associated with X‐linked Emery–Dreifuss muscle dystrophy.