蛋白质组学指南.pdf

中国试剂网 3.26.273.26.27 蛋白质组学指南 Molecular Biologists Guide to Proteomics Paul R. Graves1 and Timothy A. J. Haystead1,2* Department of Pharmacology and Cancer Biology, Duke University,1,1 and Serenex Inc.,2 Durham, North Carolina 277102 Microbiology and Molecular Biology Reviews, March 2002, p. 39-63, Vol. 66, No. 1 This review is intended to give the molecular biologist a rudimentary understanding of the technologies behind proteomics and their application to address biological questions. Entry of our laboratory into proteomics 5 years ago was driven by a need to define a complex mixture of proteins ( 36 proteins) we had affinity isolated that bound specifically to the catalytic subunit of protein phosphatase 1 (PP-1, a serine/threonine protein phosphatase that regulates multiple dephosphorylation events in cells) (26). We were faced with the task of trying to understand the significance of these proteins, and the only obvious way to begin to do this was to identify them by sequencing. We then bought an Applied Biosystems automated Edman sequencer (not having the budget for a mass spectrometer at the time). Since the majority of intact eukaryotic proteins are not immediately accessible to Edman sequencing due to posttranslational N-terminal modifications, we invented mixed-peptide sequencing (38). This method, described in detail later, essentially enables internal peptide sequence information to be derived from proteins electroblotted onto hydrophobic membranes. Using the mixed-peptide sequencing strategy, we identified all 36 proteins in about a week. The mixture contained at least two known PP-1 regulatory subunits, but most were identified in