TANDEM MASS-SPECTROMETRIC CHARACTERIZATION OF A SPECIFIC CYSTEIC ACIDRESIDUE IN OXIDIZED HUMAN APOPROTEIN B-100

The oxidation of low density lipoprotein (LDL) in vivo may result in i ts unregulated uptake by macrophages, with the consequent accumulation of cholesterol that is characteristic of the development of atheroscl erosis. This paper describes initial experiments to elucidate structur al changes that occur in an in vitro model of LDL oxidation. LDL was i solated from human blood and oxidized in the presence of copper ion. L ipid was removed and the isolated apoprotein was subjected to tryptic hydrolysis. The hydrolysate was separated by high performance liquid c hromatography and individual fractions were screened by amino acid ana lysis to detect cysteic acid residues. Appropriate fractions were anal yzed by fast atom bombardment mass spectrometry and hybrid tandem mass spectrometry. In this manner a tryptic fragment was identified that c orresponded to residues 4187-4195 (EELCTMFIR), in which the cysteine a nd methionine residues were oxidized to cysteic acid and methionine su lfoxide, respectively. Identical analysis of LDL not subjected to in v itro oxidation revealed no evidence for this oxidized peptide. Earlier work established a surface location for this cysteine residue (Cys24) on the LDL particle, which suggested that its modification may signif icantly affect the properties of LDL, such as;the propensity to interm olecular interaction via disulfide bridges. The analytical protocol de veloped here (involving proteolysis, screening of peptide fragments, a nd tandem mass spectrometry analysis) constitutes a strategy of genera l applicability to the characterization of targeted modifications of l arge proteins via mass spectrometry.