Tandem mass spectrometric characterization of a specific cysteic acid residue in oxidized human apoprotein B-100.

The oxidation of low density lipoprotein (LDL) in vivo may result in its unregulated uptake by macrophages, with the consequent accumulation of cholesterol that is characteristic of the development of atherosclerosis. This paper describes initial experiments to elucidate structural changes that occur in an in vitro model of LDL oxidation. LDL was isolated from human blood and oxidized in the presence of copper ion. Lipid was removed and the isolated apoprotein was subjected to tryptic hydrolysis. The hydrolysate was separated by high performance liquid chromatography and individual fractions were screened by amino acid analysis to detect cysteic acid residues. Appropriate fractions were analyzed by fast atom bombardment mass spectrometry and hybrid tandem mass spectrometry. In this manner a tryptic fragment was identified that corresponded to residues 4187-4195 (EELCTMFIR), in which the cysteine and methionine residues were oxidized to cysteic acid and methionine sulfoxide, respectively. Identical analysis of LDL not subjected to in vitro 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 significantly affect the properties of LDL, such as the propensity to intermolecular interaction via disulfide bridges. The analytical protocol developed here (involving proteolysis, screening of peptide fragments, and tandem mass spectrometry analysis) constitutes a strategy of general applicability to the characterization of targeted modifications of large proteins via mass spectrometry.