A gel-based reference map of the porcine hepatocyte proteome

Authors: Caperna, Thomas; Shannon, Amy; Garrett, Wesley

Submitted to: Domestic Animal Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/2007
Publication Date: 8/1/2008
Citation: Caperna, T.J., Shannon, A., Garrett, W.M. 2008. A gel-based reference map of the porcine hepatocyte proteome. Domestic Animal Endocrinology. 35(1):142-156.

Interpretive Summary: The overall goal of our research is to characterize and identify gene expression profiles of porcine hepatic cells. In particular, this report focuses on identification of the major proteins that could be identified in pig hepatocytes. We have prepared two dimensional electrophoresis maps of proteins extracted from freshly isolated hepatocytes which were pooled from three crossbred pigs (35-69 kg). Following isoelectric focusing with three different pH-range immobilized pH gradient strips (pH 3-6, 5-8 and 7-10), individual second dimension gels were run to provide six maps, from which 728 protein spots were visualized, picked and digested with trypsin. Two different mass spectrometry methods were used to identify the porcine proteins from the resulting tryptic peptides. First, proteins were identified by matrix -assisted laser desorption/ionization-time of flight mass spectrometry (MS; 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 (MS/MS). Utilizing publicly-available databases, 648 proteins were identified. Of those, 284 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.

Technical Abstract: 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 (35-69 kg). 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 (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, 284 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.