期刊
DOMESTIC ANIMAL ENDOCRINOLOGY
卷 35, 期 2, 页码 142-156出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.domaniend.2007.12.004
关键词
pig hepatocytes; 2D-PAGE; MALDI-TOF-MS; LCMS/MS
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 immnobilized 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. Published by Elsevier Inc.
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