Akhilesh Pandey Johns Hopkins University, Baltimore, MD Office: 410-502-6662 FAX: 410-502-7543 E-mail: pandey@jhmi.edu Job Title: Assistant Professor M.D. in Internal Medicine from Armed Forces Medical College, Pune, India Ph.D. in Pathology from the University of Michigan

Speaker: Akhilesh Pandey, Department of Biochemistry, Johns Hopkins University, Baltimore, MD

Topic: Proteomics Approaches to Study Signal Transduction Pathways

Place: Building 549, Auditorium, NCI at Frederick, Frederick, MD

Time: Tuesday, November 23, 2004, at 2:00 PM

Abstract: Global identification of phosphorylated proteins remains a difficult task in spite of a number of technological advances in enrichment methods and mass spectrometry. It is difficult to visualize the phosphoproteome because of the low stoichiometry of phosphorylation, low abundance of most signaling molecules and initiation of phosphorylation events only in response to specific stimuli. We have previously developed stable isotope labeling with amino acids in cell culture (SILAC) as an in vivo labeling method for relative quantitation by mass spectrometry. We have recently applied the SILAC method for specific identification of tyrosine kinase substrates. For this purpose, we labeled HeLa cells with stable isotopes of tyrosine or a combination of arginine plus lysine amino acids to identify phosphorylated proteins and their phosphorylation sites. The extent of phosphorylation was enhanced by treating cells with sodium pervanadate, an inhibitor of tyrosine phosphatases, and this differential phosphorylation was used to identify the tyrosine phosphorylated proteins. We identified 115 potentially tyrosine phosphorylated proteins, of which 72 proteins were not previously described in literature as phosphoproteins. A total of 34 in vivo tyrosine phosphorylation sites were mapped, including 26 novel ones. Western blotting experiments using antiphosphotyrosine antibodies confirmed the phosphorylation status of a subset of these novel phosphoproteins in response to EGF stimulation in addition to pervanadate treatment. Because of its sensitivity and general applicability, our approach is a useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for a functional annotation of the genome.