LC-MS3 method for the analysis of Lipid Peroxidation Products (LPPs) positional isomers

Abstract

Lipid peroxidation is an enzymatic or non-enzymatic transformation of polyunsaturated fatty acids (PUFAs) leading to the formation of various lipid peroxidation products (LPPs) including low molecular weight aldehydes (i.e. 4-hydroxy-trans-2- nonenal, acrolein), truncated lipids (i.e. alkanals, alkenals), and hydroxy-alka(e)nals), isoprostanes, hydroperoxy-, hydroxy-, keto- and epoxy-derivatives. Numerous studies shown the involvement of lipid peroxidation in the onset and progression of inflammatory based diseases such as diabetes, Alzheimer, Parkinson diseas and cardiovascular diseases [1]. LPPs are known to modulate different cellular signaling pathways by inducing changes in the biological membranes [2], protein lipoxidation, and interaction with cell surface (e.g. scavenger receptors and TLRs) [3] and intracellular (e.g. PPARs) receptors [4]. Various biological activities of LPPs are mainly determined by their chemical diversity. In order to understand LPPs structure-functional relationships, specific and sensitive analytical tools allowing separation and identification of structural isomers are required. Liquid chromatography coupled on-line to mass spectrometry (LC-MS) allows high-throughput characterization of LPPs in biological samples. However, majority of the methods are not capable to distinguish LPP structural isomers. Here a novel LC-MS method for analysis of isomeric LPPs was developed. MS and tandem MS analysis was performed in negative ion mode. Anions of oxidized PUFAs, produced by collision induced dissociation of LPP precursors, were further used for data-driven MS3 analysis yielding structure specific fragment ions necessary to assign type and position of oxidation within PUFA alkyl chain. Method was validated using in vitro oxidized standard PLs and further translated for analysis of complex biological samples. Thus, using novel LC-MS3 based approach it was possible to reveal structural diversity of LPPs in a high-throughput manner.