Abstract
Oxidized phospholipids (OxPLs) are increasingly recognized as signaling mediators that are not only markers of oxidative stress but are also "makers" of pathology relevant to disease pathogenesis. Understanding the biological role of individual molecular species of OxPLs requires the knowledge of their concentration kinetics in cells and tissues. In this work, we describe a straightforward "fingerprinting" procedure for analysis of a broad spectrum of molecular species generated by oxidation of the four most abundant species of polyunsaturated phosphatidylcholines (OxPCs). The approach is based on liquid-liquid extraction followed by reversed-phase HPLC coupled to electrospray ionization MS/MS. More than 500 peaks corresponding in retention properties to polar and oxidized PCs were detected within 8 min at 99 m/z precursor values using a single diagnostic product ion in extracts from human dermal fibroblasts. Two hundred seventeen of these peaks were fluence-dependently and statistically significantly increased upon exposure of cells to UVA irradiation, suggesting that these are genuine oxidized or oxidatively fragmented species. This method of semitargeted lipidomic analysis may serve as a simple first step for characterization of specific "signatures" of OxPCs produced by different types of oxidative stress in order to select the most informative peaks for identification of their molecular structure and biological role.