11 months ago

2007 Scientific Report

  • Text
  • Report
  • Institute
  • Protein
  • Signaling
  • Tumor
  • Michigan
  • Molecular


VARI | 2007 Gregory S. Cavey, B.S. Laboratory of Mass Spectrometry and Proteomics Mr. Cavey received his B.S. degree from Michigan State University in 1990. Prior to joining VARI he was employed at Pharmacia in Kalamazoo, Michigan, for nearly 15 years. As a member of a biotechnology development unit, he was group leader for a protein characterization core laboratory. More recently as a research scientist, he was principal in the establishment and application of a state-of-the-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002. 13 Staff Laboratory Staff Paula Davidson, M.S. Joan Krilich, B.S. Students Visiting Scientists

Van Andel Research Institute | Scientific Report Research Interests The Mass Spectrometry and Proteomics laboratory provides protein identification analysis and protein molecular weight determination as core services. Nanogram amounts of protein in SDS-PAGE gels or in solution are digested into peptides and analyzed by HPLC with on-line electrospray mass spectrometry. Peptides are fragmented in the mass spectrometer to generate amino acid sequence data that is used to identify proteins by searching protein and DNA databases. Submicrogram amounts of intact proteins are analyzed by nanoscale liquid chromatography–mass spectrometry (LC-MS) to determine their average molecular weight; this work is performed using a variety of HPLC columns to optimize recovery and provide reliable results. These core services are provided to both VARI investigators and external clients. Research in the lab focuses on improving existing services and developing new methods based on the needs of VARI investigators. Our three main areas of interest are intact-protein molecular weight determination, phosphopeptide analysis, and protein expression profiling using LC-MS. Protein LC-MS 14 We use protein LC-MS to confirm correct expression and purification of recombinant proteins from bacteria. The average molecular weight of a protein is experimentally determined and compared with the calculated weight from the expected amino acid sequence. Proteins of 50 kDa and larger are analyzed with mass accuracy often better than 0.01%, or ±1 Da per 10 kDa. Unlike with conventional SDS-PAGE, protein truncation and modifications such as oxidation or acetylation can be accurately characterized using protein LC-MS. This information is essential when protein reagents are used for labor-intensive and costly protocols such as x-ray crystallography, antibody production, or drug screening. We have a dedicated LC-MS instrument with optimized HPLC separation and comprehensive data processing for analyzing complex mixtures of proteins. For proteins that degrade during purification, we can alter the use of protease inhibitors or minimize degradation through site-directed mutagenesis of susceptible amino acids. We are also exploring the use of this equipment for biomarker discovery of intact proteins. The goal is to provide relative quantitation of proteins in disease cell culture models, tumor tissue, and cancer patient body fluids. Protein phosphorylation analysis Mapping post-translational modifications of proteins such as phosphorylation is an important yet difficult undertaking in cancer research. Phosphorylation regulates many protein pathways that could serve as potential drug targets in cancer therapy. In recent years, mass spectrometry has emerged as a primary tool in determining site-specific phosphorylation and relative quantitation. Phosphorylation analysis is complicated by many factors, but principally by the low-stoichiometry modifications that may regulate pathways: we are sometimes dealing with 0.01% or less of phosphorylated protein among a large excess of a nonphosphorylated counterpart. Our lab collaborates with investigators to map protein phosphorylation using techniques including multiple enzyme digestion, titanium dioxide phosphopeptide enrichment, and phosphorylation-specific mass spectrometry detection. Although trypsin is often the enzyme of choice for digesting proteins into peptides for identification, additional enzymes such as Lys-C, Staph V8, chymotrypsin, thermolysin, or elastase may also be employed. Multiple enzyme digests and titanium dioxide enrichment are used in combination with precursor ion scanning for –79 m/z on a Waters Q-Tof Premier mass spectrometer.

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