Abstract 428: Peptides Enrichment by Hydrophobic Interaction Chromatography for Detection of Oxidized Phospholipid-modified Protein Adducts

Abstract

Oxidized phospholipids (oxPLs) accumulate at sites of oxidative stress and contribute significantly to atherosclerosis, acute inflammation, lung injury, and ischemia. Most oxPLs have electrophilic substituents and are highly likely to form covalent adducts with proteins, thus compromising protein function. Detection of covalent interaction between oxPLs and proteins could provide important information about the type of proteins preferentially modified by oxPLs and on the mechanism of interaction between proteins and oxPLs. These studies are very challenging due to low levels of oxPLs protein adducts. We have developed a highly efficient enrichment method for oxPLs-modified peptides that employs C18 solid phase extraction columns. This method takes advantage of the difference in polarity between oxPLs modified peptides and the corresponding unmodified peptides as well as other interfering compounds. Using this enrichment method, we have been able to detect very low levels of oxPLs modified peptides (< 2 ppm) from tryptic digestion mixture of cell lysate using LC-MS. To demonstrate the performance of the method, we studied the interaction of apolipoprotein A-I (apoA-I) in high density lipoprotein with a specific oxPL (1-palmitoyl-2-(5’-oxo-valeroyl)-sn-glycero-3-phosphocholine, POVPC) at pathophysiological concentrations. We found that Lys 94, Lys 96 and Lys 195 are covalently modified by POVPC. Thus, the present study is the first to detected oxPL adducts of apoA-I in HDL.