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.

Keywords: LC-MS; lipidomic; nitration; nitroxidative stress; phosphatidylserine; tandem mass spectrometry.

Figure 1

ESI-MS spectrum of POPS after nitration reaction acquired in the negative-ion mode in Q-Exactive Orbitrap. Assignments of nitrated and nitroxidized derivatives formed after reaction between NO₂BF₄ and POPS observed in the ESI-MS spectrum as [M − H]⁻ ions were confirmed by mass accuracy. The calculated and observed mass, error, and formula of the nitrated and nitroxidized derivatives formed after reaction between NO₂BF₄ and POPS observed in the ESI-MS spectrum are also shown. Error (ppm) = (Observed m/z − Calculated m/z)/Calculated m/z) × 1 × 10⁶.

Figure 2

ESI-MS/MS spectra obtained in Q-Exactive Orbitrap of [M − H]⁻ ions of nitroso, nitrated and nitroxidized POPS at m/z 789.5009 ([POPS + 29 u − H]⁻) assigned as NO-POPS (A), at m/z 805.4980 ([POPS + 45 u − H]⁻) assigned as NO₂-POPS (B), at m/z 821.4911 ([POPS + 61 u − H]⁻) assigned as (NO₂)O-POPS (C), at m/z 834.4889 ([POPS + 74 u − H]⁻) assigned as (NO₂)(NO)-POPS (D), at m/z 837.4868 ([POPS + 77 u − H]⁻) assigned as (NO₂)2O-POPS (E), and at m/z 850.4836 ([POPS + 90 u − H]⁻) assigned as (NO₂)₂-POPS (F). One possible chemical structure of each deprotonated molecule is also proposed, with the nitro group located in C9, but other possibilities should be considered, namely the nitro group at C10, as reported for the nitration of oleic acid [12].

Figure 3

Reconstructed ion chromatogram (RIC) and LC-MS/MS spectra (A–G) of nitrated and nitroxidized derivatives of POPS at m/z 789 identified as nitroso POPS (RT 20.47 min) (A), isobaric compounds at m/z 805 identified as nitro (RT 28.92) (B) and nitrosohydroxy POPS (20.44 min) (C), at m/z 821 identified as nitrohydroxy POPS (RT 16.68 min) (D), at m/z 834 identified as nitronitroso POPS (RT 17.38 min) (E), at m/z 837 identified as nitrohydroperoxy POPS (RT 18.30 min) (F), and at m/z 850 identified as dinitro POPS (RT 19.85 min) (G).

Figure 4

Schematic representation of major fragmentation pathways of nitrated POPS derivatives observed in negative-ion mode. A possible chemical structure of nitrated POPS with the nitro (NO₂) and nitroso (NO) groups located in C9 is represented, but other possibilities should be considered, namely the nitro (NO₂) and nitroso (NO) groups at C10, as reported for the nitration of oleic acid [12].

Figure 5

Percentage of inhibition of DPPH^● and ABTS^●+ radicals remaining after 120 min of reaction in the presence of nitrated POPS at the three concentrations tested (37.5, 75 and 150 µg/mL in ethanol).