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Nitrated phospholipids have recently been detected in vitro and in vivo and associated with beneficial health effects. They were identified and quantified in biological samples by lipidomics methodologies using liquid chromatography-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) acquired with the linear ion trap mass spectrometer. Only a few studies have used higher-energy collision dissociation (HCD)-MS/MS in high-resolution Orbitraps to characterize nitrated phosphatidylserines and nitrated cardiolipins, highlighting the marked differences in the fragmentation patterns when using CID or HCD fragmentation methods. In this study, we aimed to evaluate the fragmentation of nitrated phosphatidylcholine and nitrated phosphatidylethanolamine species under HCD-MS/MS. We studied the effect of normalized collision energy (NCE) in the fragmentation pattern to identify the best acquisition conditions and reporter ions to detect nitrated phospholipids. The results showed that the intensity of the typical neutral loss of nitrous acid (HNO 2 ) diminishes with increasing NCE, becoming non-detectable for a higher NCE. Thus, the loss of HNO 2 could not be the most suitable ion/fragment for the characterization of nitrated phospholipids under HCD. In HCD-MS/MS new fragment ions were identified, corresponding to the nitrated fatty acyl chains, NO 2 -RCOO - , (NO 2 -RCOOH-H 2 O + H) + , and (NO 2 -RCOOH + H) + , suggested as potential reporter ions to detect nitrated phospholipids when using the HCD-MS/MS lipidomics analysis.

Advancing Target Identification of Nitrated Phospholipids in Biological Systems by HCD Specific Fragmentation Fingerprinting in Orbitrap Platforms