Proteomics of Macrophage: An Approach to Understand Lipopolysaccharide-challenged Inflammation

Abstract

Lipopolysaccharide (LPS) has been implicated as one of the major cause of inflammation, sepsis and shock, that are, life-threatening syndromes often occurring in intensive care unit patients. In order to detect diseaserelevant process and biomarkers for alleviating the inflammation or sepsis shock upon bacterial infection, proteome of macrophage cell line RAW 264.7 primed with LPS for 24 hours in vitro was determined using 2DE. Isoelectric focusing was performed on whole cell extracts using 11cm pH 4 –7 IPG strips. Focused proteins were resolved in 12% SDS-polyacrylamide gels and visualized by silver-stain. The proteomes patterns were analyzed by the Melanie III 2D analysis software. Three proteins out of approximately 720 detected were decreased in abundance as a result of the priming with LPS. These proteins were isolated and identified by comparing the masses of their tryptic digested peptides with those of the known proteins, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the SWISSPROT database. The putative identities of these proteins were mitochondrial respiratory chain enzymes, glyceraldehyde-3-phosphate dehydrogenase, putative tumor suppressor molecule, prohibitin, pyrimidine nucleotides synthesis enzymes, and UMP/CMP kinase. Many of these proteins possess important functions in inflammation. This high-resolution 2DE and protein identification technique allows one to rapidly discover new potential candidate proteins which can be used to develop targeted drugs proteins for the prevention and treatment of endotoxemia.