Profile of Phosphatidylserine Modifications under Nitroxidative Stress Conditions Using a Liquid Chromatography-Mass Spectrometry Based Approach

Abstract

Nitrated lipids have been detected in vitro and in vivo, usually associated with a protective effect. While nitrated fatty acids have been widely studied, few studies reported the nitration and nitroxidation of the phospholipid classes phosphatidylcholine, and phosphatidylethanolamine. However, no information regarding nitrated and nitroxidized phosphatidylserine can be found in the literature. This work aims to identify and characterize the nitrated and nitroxidized derivatives of 1-palmitoyl-2-oleoyl-sn-3-glycero-phosphoserine (POPS), obtained after incubation with nitronium tetrafluoroborate, by liquid chromatography (LC) coupled to mass spectrometry (MS) and tandem MS (MS/MS). Several nitrated and nitroxidized products were identified, namely, nitro, nitroso, nitronitroso, and dinitro derivatives, as well as some nitroxidized species such as nitrosohydroxy, nitrohydroxy, and nitrohydroperoxy. The fragmentation pathways identified were structure-dependent and included the loss of HNO and HNO2 for nitroso and nitro derivatives, respectively. Combined losses of PS polar head group plus HNO or HNO2 and carboxylate anions of modified fatty acyl chain were also observed. The nitrated POPS also showed antiradical potential, demonstrated by the ability to scavenge the ABTS●+ and DPPH● radicals. Overall, this in vitro model of nitration based on LC-MS/MS provided additional insights into the nitrated and nitroxidized derivatives of PS and their fragmentation fingerprinting. This information is a valuable tool for targeted analysis of these modified PS in complex biological samples, to further explore the new clues on the antioxidant potential of nitrated POPS.

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

“…Nitrated PLs include many structurally diverse lipid species, and its nitro derivatives (NO 2 -PLs) were the main products identified in biological samples, including the H9c2 cardiomyoblasts [10] and cardiac mitochondria of diabetic rats treated with streptozotocin [12]. These modified lipids showed antioxidant properties thanks to their radical scavenging potential against the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS •+ ) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH • ) [11,32] and can also act as anti-inflammatory agents by inhibiting the expression of inducible nitric oxide synthase (iNOS; at the protein level) in RAW 264.7 macrophages treated with lipopolysaccharide [32]. Moreover, nitrated POPC induced a series of downstream cellular effects in SW13/cl.2 cells, showing nitrated PLs as new potential electrophilic lipid mediators with selective actions [33].…”

“…Lipidomics has been widely applied for the large-scale characterization of lipid profiles and quantification of individual lipid molecular species using either targeted or untargeted MS-based approaches that include shotgun methods and HPLC-MS based strategies. It has also been applied in the study of nitrated PLs by using CID-MS/MS [10,12] and HCD-MS/MS approaches [11,13]. A very recent work on the characterization of nitrated POPS has revealed differences in the fragmentation patterns and reporter ions typical of these modified lipids under both CID-and HCD-MS/MS conditions [11].…”

“…It has also been applied in the study of nitrated PLs by using CID-MS/MS [10,12] and HCD-MS/MS approaches [11,13]. A very recent work on the characterization of nitrated POPS has revealed differences in the fragmentation patterns and reporter ions typical of these modified lipids under both CID-and HCD-MS/MS conditions [11]. The ion activation method and the CE used in tandem MS have been reported as a key step that determines the generation of the structural information observed in the MS/MS spectra of the individual molecular species [20].…”

“…We also studied, using HCD-MS/MS experiments, other nitrated derivatives of PCs and PEs such as dinitro (2NO 2 -PLs, +90 Da) and nitronitroso ((NO 2 )(NO)-PLs, +74 Da) derivatives, as well as with nitroxidized derivatives, such as nitro-hydroxy ((NO 2 )O-PLs, +61 Da) and nitro-hydroperoxy ((NO 2 )2O-PLs, +77 Da), previously characterized by CID-MS/MS [11]. For all of these species, we observed the same tendency described above for NO 2 -POPC and NO 2 -POPE (Tables S1-S11) and the most abundant reporter ions were attributed to the modified fatty acids.…”

“…Molecules 2020, 25, 2120; doi:10.3390/molecules25092120 www.mdpi.com/journal/molecules and nitroxidized FAs [4][5][6] have been recognized as important endogenous signalling molecules [7][8][9]. On the other hand, nitrated PLs have only been reported recently in in vitro model systems and biological samples and structurally characterized by mass spectrometry (MS) approaches [10][11][12][13]. Nitrated PCs and nitrated PEs were detected in the cardiac mitochondria of diabetic rats and cardiomyocytes under starvation using liquid chromatography (LC) coupled to a linear ion trap mass spectrometer (LIT) and were characterized by low energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) [10,12].…”