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2007 Scientific Report

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  • Protein
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  • Michigan
  • Molecular

VARI |

VARI | 2007 We have developed a robust negative-ion-mode method using nanoscale HPLC that provides specific detection of phosphopeptides below 20 fmol in the presence of 2 pmol of nonphosphorylated protein. Once detected in the negative mode, phosphopeptides are sequenced in a subsequent LC-MS analysis in the positive ion mode using accurate mass parent ion selection, a narrow retention time window, and collision energy ramping. This approach has provided a reliable and sensitive means of analyzing phosphoproteins in our laboratory. Our current focus is on applying this label-free method to studies requiring relative quantitation of phosphorylation events. Protein expression/biomarker discovery As mass spectrometry instruments and protein separation methods develop, proteomics techniques allow researchers to identify and quantitate protein samples of increasing complexity. The ultimate goal is to catalog all proteins expressed in a given cell or tissue as a means of evaluating dynamic physiological events and understanding how all proteins interact to affect a biological outcome. Traditionally this goal has been approached using 2D gel electrophoresis, image analysis of stained proteins, and identification of proteins from gels using mass spectrometry. Because of the labor-intensive nature of 2D gels and the underrepresentation of some protein classes (such as membrane proteins), proteomics has been moving toward solution-based separations and direct mass spectrometry analysis. Our laboratory recently purchased and installed a Waters Corporation Protein Expression System for non-gel-based, label-free protein expression analysis. This system represents a paradigm shift in the field of proteomics, because it provides both quantitative and qualitative data on complex mixtures of proteins in a single LC-MS analysis. Proteins are enzymatically digested using trypsin and, without any chemical or isotopic labeling, the resulting peptides are analyzed by LC-MS. The combination of molecular mass and LC retention time establishes a signature for each peptide and allows comparison across samples. The mass spectrometer signal intensity of each peptide is used for quantitation. Qualitative protein identification data is obtained by fragmenting all peptides eluting into the mass spectrometer, a feature unique to the Waters instrument. VARI is one of an elite group of institutions that have this powerful new technology. This system will be used to map protein pathways under a systems biology approach and to discover potential biomarkers for early detection and diagnosis in cancer and other diseases. 15 External Collaborators Gary Gibson, Henry Ford Hospital, Detroit, Michigan Michael Hollingsworth, Eppley Cancer Center, University of Nebraska, Omaha Waters Corporation Core Technology Alliance (CTA) This laboratory participates in the CTA as a member of the Michigan Proteomics Consortium. From left: Davidson, Cavey, Krilich

Van Andel Research Institute | Scientific Report 16 Cells prepared by Miles Qian and Daisuke Matsuda of the Teh laboratory. Image by Kristin VendenBeldt of the Resau laboratory.

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