Analysis of Phosphatidylinositol Modifications by Reactive Nitrogen Species Using LC-MS: Coming to Grips with Their Nitroxidative Susceptibility

Phosphatidylinositols(PIs) are complex lipids that play a keyrole in cell signaling. Like other phospholipids, they are esterifiedwith unsaturated fatty acyl residues (FAs), making them susceptibleto modification by reactive oxygen and nitrogen species (RNS). Recentstudies using mass spectrometry (MS)-based lipidomics approaches haverevealed that lipid nitration results in a plethora of structurallyand chemically modified lipids (epilipids), including nitrated andnitroxidized derivatives of phosphatidylcholines, phosphatidylethanolamines,phosphatidylserines, and cardiolipins. However, there is a notablelack of knowledge regarding the characterization of RNS-modified PIderivatives. In this study, we used C18 high-resolution liquid chromatography-tandemMS approaches to describe the fragmentation signature of nitratedand nitroxidized PIs, bearing different fatty acyl chains. Using thisapproach and accurate mass measurements, we were able to identifynitro- PI derivatives, dinitro- and nitrohydroxy- derivatives fora few PI species. The data showed the typical neutral loss of nitrousacid (HNO2) as well as the fragmentation patterns correspondingto modified fatty acyl chains (such as NO x -RCOO-, [M - NO x -RCOOH - H](-) and [M - NO x -RCOOH - C6H10O5 - H](-)), making it possible to identifythese epilipids. The susceptibility of PIs to nitration was also investigated,revealing that it depends exclusively on the chains of unsaturatedFAs esterified in PI, showing a higher conversion rate for those withC18:1. Overall, the knowledge gathered in this study will contributeto the precise characterization of these epilipids in complex biologicalsamples, offering new opportunities to unveil the pathophysiologicalroles of nitrated and nitroxidized PI derivatives at the cellularand tissue levels.