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2006 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Gregory S. Cavey, B.S. Laboratory of Mass Spectrometry and Proteomics 14 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-of-the-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002. Staff Laboratory Staff Paula Davidson, M.S. Student Students Wendy Johnson Visiting Scientists

VARI | 2006 Research Interests The Mass Spectrometry and Proteomics program works with many of the research labs at the Institute, using state-of-the-art mass spectrometers in combination with analytical protein separation and purification methods to help answer a wide range of biological questions. 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 hypotheses being addressed. Protein-protein interactions Analyzing 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. 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. 15 Recently, we have been evaluating newly developed software that allows us to eliminate the electrophoresis separation step 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 high-pressure liquid chromatography–mass spectrometry (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 also characterizes 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 labor-intensive 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, 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 by the fact that pathway regulation can occur when only 0.01% of a given protein population is phosphorylated. Thus, we are dealing with an extremely small number of phosphorylated proteins among a huge number of nonphosphorylated proteins. Our lab collaborates with investigators to map protein phosphorylation using a variety of techniques, including metal affinity purification, immunoaffinity purification of phosphoproteins and peptides, and phosphorylation-specific mass spectrometry detection.

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