Abstract 258: High-Efficiency Enrichment of Oxidized Phospholipid-Modified Peptides with C18 Solid Phase Extraction Column Facilitates Detection of Covalent Interaction Between Oxidized Phospholipids and Proteins
Oxidized phospholipids accumulate at sites of oxidative stress and contribute significantly to atherosclerosis, acute inflammation, lung injury and ischemia. Most of the oxidized phospholipids have electrophilic substituents and are highly likely to form covalent adducts with proteins thus compromising protein function. Detection of covalent interaction between oxidized phospholipids and proteins could provide important information about proteins preferentially modified by oxidized phospholipids in a relevant biological system, as well as, shed light on the mechanism of interaction between proteins and oxidized phospholipids. However, due to the relatively low levels of phospholipid protein adducts compared to unmodified proteins, such studies are challenging. We developed a high efficiency enrichment method for oxidized phospholipid-modified peptides that employs a C18 SPE column. Using this approach, in combination with LC-MS analysis, we studied the interaction of a synthetic oxidized phospholipid (9-keto-12-oxo-10-dodecenoic acid esters of 2-lysoPC, KODA-PC) with Apo-AI in HDL. We found that Lys45, His162, His193 and Lys208 are the major residues for covalent modification by oxidized phospholipids. According to the double superhelix model of HDL, these residues are located at both terminals of the double superhelix of Apo-AI. Thus the present study, for the first time, successfully detected oxidized phospholipids adducts of ApoA-I in HDL.
- © 2012 by American Heart Association, Inc.