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2002 Scientific Report

  • Text
  • Institute
  • Report
  • Tumors
  • Protein
  • Signaling
  • Michigan
  • Molecular
  • Proteins
  • Laboratory

Mass Spectrometry and

Mass Spectrometry and Proteomics Laboratory Gregory S. Cavey, B.S. 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 part of a biotechnology development unit, he was a group leader for a protein characterization core laboratory. More recently as a research scientist in discovery research, Greg was principal in the establishment and applications of a state-of-the-art proteomics laboratory for drug discovery. He joined VARI as a Special Program Investigator in July 2002. Research Projects P roteomics is fast becoming a major effort in most research institutions. The rapid development of technology is providing powerful new tools for probing the function of proteins and for extracting information from the Human Genome project. Since nearly all drug targets are proteins, there is clear incentive to apply proteomics as a complementary approach to genomics research. For proteomics, the most important technology is the coupling of quantitative analytical protein separation and modern mass spectrometers to identify and characterize proteins with unprecedented sensitivity and throughput. This laboratory will collaborate with VARI investigators in applying current proteomics technology toward their research goals and will build external collaborations to speed the development of new tools to meet the many challenges in cancer research. Expression proteomics and cell-mapping proteomics will be pursued, as will characterization of posttranslational modifications such as phosphorylation. For expression proteomics, we will initially rely on two-dimensional (2D) gel electrophoresis to display differentially expressed proteins of a given disease state, genetic manipulation, or drug treatment. This may require implementing an array of sample isolation, solubilization, and fractionation techniques. Once the proteins are displayed on 2D gels, quantitative data can be used to identify proteins of interest, followed by automated identification using mass spectrometry and database searching. Forthcoming will be the use of an isotopecoded affinity tagging (ICAT) approach that will allow quantitative measurement of proteins in control vs. experimental samples. This approach will require the set-up and use of a multidimensional liquid chromatography system for the separation of complex mixtures of proteins. Cell-mapping proteomics will be used to identify components of protein complexes under various conditions in order to help understand the regulatory mechanism(s) of a given pathway. In this approach, a nondenatured sample is affinity-purified using either antibodies, a known protein carrying an affinity tag, or immobilized small molecules. Binding partners are separated by 2D or SDS polyacrylamide gel electrophoresis (PAGE) and are identified using mass spectrometry and database searching. Posttranslational modification of proteins—in particular phosphorylation—is known to be a regulatory event in signal transduction. The proteomics lab will work with various investigators to map phosphorylation sites of proteins. The unique talents, research, and resources at VARI provide numerous opportunities for proteomics applications. 17

Laboratory of Signal Regulation and Cancer Sara A. Courtneidge, Ph.D. Dr. Courtneidge completed her Ph.D. at the National Institute for Medical Research in London. She began her career in the basic sciences in 1978 as a Postdoctoral Fellow in the laboratory of J. Michael Bishop at the University of California School of Medicine. She later joined her alma mater as a member of the scientific staff. In 1985 Dr. Courtneidge joined the European Molecular Biology Laboratory as Group Leader and in 1991 was appointed Senior Scientist with tenure. She joined Sugen in 1994 as Vice President of Research, later becoming Senior Vice President of Research and then Chief Scientist. Dr. Courtneidge was appointed Senior Scientific Investigator and Deputy Director of the Van Andel Research Institute in January 2001. Laboratory Members Staff Eduardo Azucena, Ph.D. Hasan Korkaya, Ph.D. Darren Seals, Ph.D. Rebecca Uzarski, Ph.D. Rebecca Cruz, M.S. Daniel Salinsky, M.S. Students Erik Freiter, B.S. Lisa Maurer Therese Roth Research Projects Our lab wants to understand at the molecular level how proliferation is controlled in normal cells and by what mechanisms these controls are subverted in tumor cells. We largely focus on a family of oncogenic tyrosine kinases, the Src family. The prototype of the family, vSrc, originally discovered as the transforming protein of Rous sarcoma virus, is a mutated and activated version of a normal cellular gene product, cSrc. The activity of all members of the Src family is normally under strict control; however, the enzymes are frequently activated or overexpressed, or both, in human tumors. In normal cells, Src family kinases have been implicated in signaling from many types of receptors, including receptor tyrosine kinases, as well as integrin receptors and G protein-coupled receptors. Signals generated by Src family kinases are thought to play a role in cell cycle entry, cytoskeletal rearrangements, cell migration, and cell division. In tumor cells, Src may play a role in growth factor–independent proliferation or in invasiveness. In addition, some evidence points to a role for Src family kinases in angiogenesis. Some of the current projects in the laboratory are outlined below. Novel Src substrates We recently described a method for identifying tyrosine kinase substrates by using anti-phosphotyrosine antibodies to screen tyrosine-phosphorylated cDNA expression libraries. Several potential Src substrates were identified, including Fish, which has five SH3 domains and a phox Src-transformed cells were stained to visualize Fish (green) and F-actin (red). The podosomes are visible as rings of intense F-actin staining. Much of Fish is also present in these podosomes. 18

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