Capture of proteins in rat liver by affinity chromatography using immobilized lithocholic acid and identification with liquid chromatography-mass spectrometry

Abstract

Affinity chromatography is a powerful method for protein separation. It is based on a specific interaction between an immobilized ligand and the target proteins to be separated. Since lithocholic acid (LCA), one of secondary bile acids, has been shown to exert its function as the ligand toward nuclear receptors and a membrane-type G protein-coupled receptor, the abilities of molecular recognition and acquisition of LCA may be applicable for ligands to capture unknown functional proteins by affinity chromatography. In this study, LCA was covalently bound to an activated agarose through a bridge introduced at the C-3 and C-24 positions. The affinity absorbents were applied to capture proteins in a rat mitochondrial fraction. Structure analysis of the captured proteins after SDS-PAGE separation was carried out by liquid chromatography/electrospray ionization-tandem mass spectrometry combination with computer-assisted programs, where carbamoyl phosphate synthase I, glutamate dehydrogenase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, acetyl-CoA acyltransferase and aldehyde dehydrogenase were identified. Serum albumin and cytosolic glutathione S-transferase, which were contaminated in mitochondrial fraction, were also identified. © 2007 The Japan Society for Analytical Chemistry.