Simultaneous Detection of Low and High Molecular Weight Carbonylated Compounds Derived from Lipid Peroxidation by Electrospray Ionization-Tandem Mass Spectrometry

Reactive oxygen species (ROS) and other oxidative agents such as free radicals can oxidize polyunsaturated fatty acids (PUFA) as well as PUFA in lipids. The oxidation products can undergo consecutive reactions including oxidative cleavages to yield a chemically diverse group of products, such as lipid peroxidation products (LPP). Among them are aldehydes and ketones (“reactive carbonyls”) that are strong electrophiles and thus can readily react with nucleophilic side chains of proteins, which can alter the protein structure, function, cellular distribution, and antigenicity. Here, we report a novel technique to specifically derivatize both low molecular and high molecular weight carbonylated LPP with 7-(diethylamino)­coumarin-3-carbohydrazide (CHH) and analyze all compounds by electrospray ionization-mass spectrometry (ESI-MS) in positive ion mode. CHH-derivatized compounds were identified by specific neutral losses or fragment ions. The fragment ion spectra displayed additional signals that allowed unambiguous identification of the lipid, fatty acids, cleavage sites, and oxidative modifications. Oxidation of docosahexaenoic (DHA, 22:6), arachidonic (AA, 20:4), linoleic (LA, 18:2), and oleic acids (OA, 18:1) yielded 69 aliphatic carbonyls, whose structures were all deduced from the tandem mass spectra. When four phosphatidylcholine (PC) vesicles containing the aforementioned unsaturated fatty acids were oxidized, we were able to deduce the structures of 122 carbonylated compounds from the tandem mass spectra of a single shotgun analysis acquired within 15 min. The high sensitivity (LOD ∼ 1 nmol/L for 4-hydroxy-2-nonenal, HNE) and a linear range of more than 3 orders of magnitude (10 nmol/L to 10 μmol/L for HNE) will allow further studies on complex biological samples including plasma

Recent Advances on Mass Spectrometry Analysis of Nitrated Phospholipids

In recent years, there has been an increasing interest in nitro fatty acids (NO₂-FA) as signaling molecules formed under nitroxidative stress. NO₂-FA were detected <i>in vivo</i> in a free form, although it is assumed that they may also be esterified to phospholipids (PL). Nevertheless, insufficient discussion about the nature, origin, or role of nitro phospholipids (NO₂-PL) was reported up to now. The aim of this study was to develop a mass spectrometry (MS) based approach which allows identifying nitroalkenes derivatives of three major PL classes found in living systems: phosphatidylcholines (PCs), phosphatidylethanolamine (PEs), and phosphatidylserines (PSs). NO₂-PLs were generated by NO₂BF₄ in hydrophobic environment, mimicking biological systems. The NO₂-PLs were then detected by electrospray ionization (ESI-MS) and ESI-MS coupled to hydrophilic interaction liquid chromatography (HILIC). Identified NO₂-PLs were further analyzed by tandem MS in positive (as [M + H]⁺ ions for all PL classes) and negative-ion mode (as [M – H]⁻ ions for PEs and PSs and [M + OAc]⁻ ions for PCs). Typical MS/MS fragmentation pattern of all NO₂-PL included a neutral loss of HNO₂, product ions arising from the combined loss of polar headgroup and HNO₂, [NO₂-FA + H]⁺ and [NO₂-FA – H]⁻ product ions, and cleavages on the fatty acid backbone near the nitro group, allowing its localization within the FA akyl chain. Developed MS method was used to identify NO₂-PL in cardiac mitochondria from a well-characterized animal model of type 1 diabetes mellitus. We identified nine NO₂-PCs and one NO₂-PE species. The physiological relevance of these findings is still unknown