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2004 Scientific Report

Laboratory of Mass

Laboratory of Mass Spectrometry and Proteomics 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 a member of a biotechnology development unit, he was group leader for a protein characterization core laboratory. More recently as a research scientist in discovery research, he was principal in the establishment and application of a state-ofthe-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002. Staff Veronica Mutchler, B.A. Laboratory Members Students Wendy Schroeder Marie Graves Research Interests T he Mass Spectrometry and Proteomics program works with many of the research labs at the Institute to help answer a wide range of biological questions. State-of-the-art mass spectrometers, used in combination with analytical protein separation and purification methods, are powerful tools for studying proteins in disease processes. Using mass spectrometry data and database search software, proteins can be identified and characterized with unprecedented sensitivity and throughput. Since proteomics is a relatively new scientific discipline, many of the analytical techniques are rapidly changing; therefore our mission involves using established protocols, improving them, and developing new approaches to expand the scope of biological challenges being addressed. Protein-protein interactions Evaluating samples representing different cellular conditions or disease states is a step toward understanding the role of a protein with an unknown function or understanding the regulatory mechanism of several proteins in a given pathway. In this approach, a known protein is affinity-purified from a nondenatured sample using antibodies, affinity tags such as FLAG or TAP, or immobilized small molecules. The purified protein and its binding partners are separated using two-dimensional (2D) electrophoresis gels or SDS-PAGE. After staining, the proteins are cut from the gel, digested into peptides using an enzyme such as trypsin, and then analyzed by nanoscale high-pressure liquid chromatography on line with a mass spectrometer (LC-MS). The mass spectrometer fragments the peptides and the resulting spectra are used to search protein or translated DNA databases. Identifications are made using the amino acid sequences derived from the mass spectrometry data. We have optimized all aspects of this analysis for sample recovery yields and high-sensitivity protein identification. Recently, we have been evaluating newly developed software that allows the electrophoresis separation step to be eliminated from these analyses, giving the potential to identify more proteins from complex mixtures. With this software, affinity-purified protein complexes are compared to a control sample using a technique known as peptide differential display. The proteins are digested into peptides in solution rather than from gels and are analyzed by LC-MS. Peptides that are unique to the experimental sample relative to the control are used to identify proteins that are part of a protein complex. Protein characterization Our laboratory supports investigators by characterizing proteins and their post-translational modifications. Proteins expressed and purified by investigators are analyzed by protein electrospray to confirm the average protein molecular weight before proceeding to laborintensive studies such as protein crystallization. Mapping the post-translational modifications of proteins such as phosphorylation is an important yet difficult undertaking in cancer research. Phosphorylation regulates many protein pathways, several of which could serve as potential drug targets for cancer therapy. In recent years, 16

mass spectrometry has emerged as a primary tool that helps investigators determine exactly which amino acids of a protein are modified. This undertaking is complicated by many factors, but principally because pathway regulation can occur when only 0.01% of the molecules of a given protein are phosphorylated. Thus, we are dealing with an extremely small number of molecules, in addition to the fact that the purification of phosphopeptides is always difficult. Our lab collaborates with investigators to map protein phosphorylation using a variety of techniques, including immobilized metal affinity purification following esterification, immunoaffinity purification of phosphoproteins and peptides, and phosphorylation-specific mass spectrometry detection. Protein expression As mass spectrometry instruments and protein separation methods develop, we hope to use proteomics research to identify and quantitate all the proteins expressed in a given cell or tissue, as a means of evaluating all of the physiological processes occurring within. This approach, termed systems biology, aims at understanding how all proteins interact to affect a biological outcome. Traditionally this has been done using 2D gel electrophoresis, image analysis of stained proteins, and identification of proteins from gels using mass spectrometry. Due to the labor-intensive nature of 2D gels and the underrepresentation of many different classes of proteins (such as membrane proteins), the field of proteomics has been moving toward solution-based separations and direct mass spectrometry. Our first approach is to digest all proteins into peptides and label the C-terminus of experimental samples with 18 O water to effect a mass shift. Experimental and control samples are then mixed and separated by multidimensional highpressure liquid chromatography using strongcation ion exchange and reverse-phase separation modes. Peptides that are differentially expressed in experimental and control samples according their 16 O/ 18 O ratio are identified using mass spectrometry and database searching. We intend to apply this or other mass spectrometry–based approaches in the discovery of biomarkers for early cancer detection, more specific diagnosis, and more accurate prognosis following drug treatment. We are collaborating with Craig Webb’s lab in developing expression analysis methods. External Collaborators Gary Gibson, Henry Ford Hospital, Detroit, Michigan Peter Leopold, BioAnalyte Inc., Portland, Maine Brett Phinney, Michigan State University, East Lansing From left to right: Mutchler, Schroeder, Graves, Cavey 17

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