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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report As with most mass spectrometry–based methods, the mapping of phosphorylation sites on proteins begins by enzymatically digesting protein into peptides using trypsin, Lys-C, Staph V8, or chymotrypsin. Peptides are separated by nanoscale reversephase HPLC and analyzed by on-line electrospray ionization on a Q-Tof mass spectrometer. Samples are analyzed using MS E data acquisition. MS E toggles the collision energy in the mass spectrometer between high and low every second throughout the analytic run. Low-collision-energy data acquisition allows peptide mass to be recorded at high sensitivity with high mass accuracy to implicate phosphorylation based on mass alone. The peptide intensity measured in the mass spectrometer is also recorded and used for relative quantitation in time course studies. During high-collision-energy acquisition, all peptides are fragmented to identify the protein(s) that the peptides were liberated from and to locate specific phosphorylated amino acids. MS E differs from other mass spectrometry approaches because fragmentation occurs for all peptides, not just for the most abundant peptides. We recently used this method for mapping phosphorylation sites on RhoA and RhoC following in vitro phosphorylation by protein kinase C epsilon (PKCe). We are currently analyzing RhoA and RhoC in a head and neck squamous cell carcinoma tissue culture model with or without the expression of PKCe using siRNA knock-down. External Collaborators Gary Gibson, Henry Ford Hospital, Detroit, Michigan Quintin Pan, Ohio State University Comprehensive Cancer Center Waters Corporation Recent Publications Yang, Maozhou, Xinli Wang, Liang Zhang, Chiyang Yu, Bingbing Zhang, William Cole, Greg Cavey, Paula Davidson, and Gary Gibson. 2008. Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag. Journal of Chromatography B 875(2): 493–501. From left to right, standing: Cavey, Lehner, Davidson; seated: Guthrey, Welsh 16

VARI | 2009 Nicholas S. Duesbery, Ph.D. Laboratory of Cancer and Developmental Cell Biology Nick Duesbery received a B.Sc. (Hon.) in biology (1987) from Queen’s University, Canada, and both his M.Sc. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada, under the supervision of Yoshio Masui. Before his appointment as a Scientific Investigator at VARI in April 1999, he was a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute, Frederick Cancer Research and Development Center, Maryland. Dr. Duesbery was promoted to Senior Scientific Investigator and appointed Deputy Director for Research Operations in 2006. Staff Students Visiting Scientist Jennifer Bromberg-White, Ph.D. Jaclyn Lynem, B.S. Elissa Boguslawski Laura Holman Chih-Shia Lee, M.S. Danielle Hawkins, B.S. Emily Olenzek, B.S. Michelle Dawes Shannon Moran Roe Froman, D.V.M. 17

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