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Recent Advances on Mass Spectrometry Analysis of Nitrated Phospholipids

Melo, Tânia, Domingues, Pedro, Ferreira, Rita, Milic, Ivana, Fedorova, Maria, Santos, Sérgio M., Segundo, Marcela A., Domingues, M. Rosário M.
Analytical chemistry 2016 v.88 no.5 pp. 2622-2629
animal models, electrospray ionization mass spectrometry, esterification, fatty acids, hydrophilic interaction chromatography, hydrophobicity, insulin-dependent diabetes mellitus, ionization, ions, mitochondria, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines
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 in vivo 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.