11 months ago

2008 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report As with most mass spectrometry–based methods, mapping phosphorylation sites on proteins begins by enzymatically digesting protein into peptides using trypsin, Lys-C, Staph V8, or chymotrypsin. Peptides are separated by nanoscale reverse-phase HPLC and analyzed by on-line electrospray ionization on a quadrupole time-of-flight (Q-Tof) mass spectrometer. Samples are analyzed using the MS E data acquisition mentioned above. 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) from which the peptides were liberated by enzyme digestion 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 are currently using this method on several in vitro phosphorylation projects, but our goal is to extend these analyses to in vivo systems to identify novel kinase or phosphatase substrates. External Collaborators Gary Gibson, Henry Ford Hospital, Detroit, Michigan Michael Hollingsworth, Eppley Cancer Center, University of Nebraska, Omaha Waters Corporation Core Technology Alliance (CTA) This laboratory participates in the CTA as a member of the Michigan Proteomics Consortium. From left: Cavey, Lehner, Davidson 14

VARI | 2008 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 Jennifer Bromberg-White, Ph.D. Philippe Depeille, Ph.D. Yan Ding, Ph.D. John Young, M.S. Jaclyn Lynem, B.S. Elissa Boguslawski Laura Holman Students Chih-Shia Lee, M.S. Naomi Asantewa-Sechereh Michelle Dawes Lisa Orcasitas Jennifer Wilcox 15

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