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2005 Scientific Report

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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 stateof-the-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002. Staff Paula Davidson, M.S. Laboratory Members Student Wendy Johnson Research Interests The Mass Spectrometry and Proteomics program works with many of the research labs at the Institute, using stateof-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 challenges 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 affinitypurified from a nondenatured sample. 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, enzymatically digested into peptides, 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 us to eliminate the electrophoresis separation step from these analyses, giving the potential to identify more proteins from complex mixtures. With this software, affinitypurified protein complexes are compared to a control sample via peptide differential display. The proteins are digested into peptides in solution rather than from gels and are analyzed by LC-MS. Peptides 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. Purified proteins 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 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 the molecules of a given protein are 16

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 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 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 approach has used 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 some classes of proteins (such as membrane proteins), proteomics has been moving toward solution-based separations and direct mass spectrometry. Our first approach is to digest all proteins into peptides and label their C-terminus with 18 O water to effect a mass shift. Experimental and control samples are then mixed and separated by multidimensional high-pressure liquid chromatography using strong-cation ion exchange and reverse-phase separation modes. Peptides that are differentially expressed in experimental and control samples according to 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, for morespecific diagnosis, and for more-accurate prognosis following drug treatment. External Collaborators Greg Fraley, Hope College, Holland, Michigan Gary Gibson, Henry Ford Hospital, Detroit, Michigan Brett Phinney, Michigan State University, East Lansing Recent Publications Li, Yong, Mihwa Choi, Greg Cavey, Jennifer Daugherty, Kelly Suino, Amanda Kovach, Nathan C. Bingham, Steven A. Kliewer, and H. Eric Xu. 2005. Crystallographic identification and functional characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1. Molecular Cell 17(4): 491–502. From left to right: Davidson, Johnson, Cavey 17

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