2008 Scientific Report

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
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