Analysis of Covalent Modifications of Proteins by Oxidized Phospholipids Using a Novel Method of Peptide Enrichment

Free radical-induced oxidation of phospholipids contributes significantly to pathologies associated with inflammation and oxidative stress. Detection of covalent interaction between oxidized phospholipids (oxPL) and proteins by LC-MS/MS could provide valuable information about the molecular mechanisms of oxPL effects. However, such studies are very limited because of significant challenges in detection of the comparatively low levels of oxPL–protein adducts in complex biological systems. Current approaches have several limitations, most important of which is the inability to detect protein modifications by naturally occurring oxPL. We now report, for the first time, an enrichment method that can be applied to the global analysis of protein adducts with various naturally occurring oxPL in relevant biological systems. This method exploits intrinsic properties of peptides modified by oxPL, allowing highly efficient enrichment of oxPL-modified peptides from biological samples. Very low levels of oxPL–protein adducts (