A gel-based reference map of the porcine hepatocyte proteome
2008
Caperna, Thomas J. | Shannon, Amy E. | Garrett, Wesley M.
The overall goal of our research is to characterize and identify gene expression profiles of porcine hepatic cells. In this study, we have prepared two-dimensional electrophoresis maps of cytosol and membrane fractions from freshly prepared hepatocytes which were pooled from three crossbred pigs (35-69kg). Following isoelectric focusing with three pH range immobilized pH gradient strips (pH 3-6, 5-8 and 7-10) and staining the second dimension gels with colloidal Coomassie blue, 728 protein spots were picked and digested with trypsin. Extracted tryptic peptides were initially subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis for identification of proteins by peptide mass fingerprinting (PMF). Proteins which were not identified by PMF were analyzed by liquid chromatography-tandem MS. Utilizing publicly available databases [NCBInr, Swiss Prot and expressed sequence tags (EST)], 648 proteins were identified. Of those, 282 were unique proteins and greater than 90% of proteins spots contained single proteins. These data represent the first comprehensive proteomic analysis of porcine hepatocytes and will provide a database for future investigations of endocrine regulation of gene expression and metabolic processes in vitro.
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