title: Characterization of phospholipid nitroxidation by LC-MS in biomimetic models and in H9c2 Myoblast using a lipidomic approach.
abstract: Under nitroxidative stress conditions, lipids are prone to be modified by reaction with reactive nitrogen species (RNS) and different modifications were reported to occur in fatty acids. However, in the case of phospholipids (PL) studied under nitroxidative stress conditions, only nitroalkene derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were reported when using both in vitro biomimetic conditions and in vivo model system of type 1 diabetes mellitus. Therefore, in order to further explore other nitroxidative modifications of PL, a biomimetic model of nitroxidation combined with liquid chromatography mass spectrometry (MS) and MS/MS approaches were used to characterize the nitrated and nitroxidized derivatives of PCs and PEs. Single and multiple nitrated derivatives of phospholipids (PLs) such as nitroso and dinitroso, nitro, dinitro, and nitronitroso derivatives, together with nitroxidized derivatives were identified. Further, the specific MS/MS fragmentation pathways of these products were studied. Product ions arising from loss of HNO and HNO2, from the combined loss of HNO (or HNO2) and polar head groups, [NOn-FA+On+H]+and [NOn-FA+On-H]-(n=1-2) product ions corresponding to the modified fatty acyl chains were observed, depending on each modification. The knowledge obtained from the study of the MS/MS fragmentation pattern has allowed us to identify nitrated PCs, including NO2-PC, (NO2)2-PCs, (NO2)(NO)-PC, NO-PC; nitrated PEs, NO2-PEs; and nitroxidized PCs, (NO2)(2O)-PC in H9c2 cells under starvation, but not under ischemia or control conditions. The physiological relevance of this nitrated and nitroxidized PCs and PEs species observed exclusively in cardiomyoblast cells (H9c2) under starvation is still unknown but deserves to be explored.
authors.affiliation: Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal. Electronic address: taniamelo@ua.pt.
authors.forename: Tânia
authors.initials: T
authors.lastname: Melo
authors.affiliation: Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
authors.affiliation: Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal; CNC.IBILI, University of Coimbra, Coimbra, Portugal.
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Ribeiro-Rodrigues
authors.affiliation: Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal; CNC.IBILI, University of Coimbra, Coimbra, Portugal.
authors.forename: Henrique
authors.initials: H
authors.lastname: Girão
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemistry, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal.
authors.forename: Marcela A
authors.initials: MA
authors.lastname: Segundo
authors.affiliation: Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal. Electronic address: mrd@ua.pt.
authors.forename: M Rosário M
authors.initials: MRM
authors.lastname: Domingues
mesh_terms: D032701:Biomimetics; D002853:Chromatography, Liquid; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D032446:Myoblasts / Q000378:metabolism / Q000473:pathology; D010713:Phosphatidylcholines / Q000302:isolation & purification / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000302:isolation & purification / Q000378:metabolism*; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D026361:Reactive Nitrogen Species; C483858:phosphatidylethanolamine
keywords: Mass spectrometry; Nitration; Nitroxidation; Nitroxidative stress; Oxidative stress; Phosphatidylcholines; Phosphatidylethanolamines