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 18O 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 16O/18O ratio are identified using mass spectrometry and database searching.
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
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
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