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Recent Advances on Mass Spectrometry Analysis of Nitrated Phospholipids Tânia Melo,† Pedro Domingues,† Rita Ferreira,† Ivana Milic,‡,§ Maria Fedorova,‡,§ Sérgio M. Santos,∥ Marcela A. Segundo,⊥ and M. Rosário M. Domingues*,† †

Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal ‡ Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, 04109 Leipzig, Germany § Center for Biotechnology and Biomedicine, Universität Leipzig, 04109 Leipzig, Germany ∥ Department of Chemistry and CICECO, University of Aveiro, 3810-193 Aveiro, Portugal ⊥ UCIBIO, REQUIMTE, Department of Chemistry, Faculty of Pharmacy, University of Porto, 4099-002 Porto, Portugal S Supporting Information *

ABSTRACT: In recent years, there has been an increasing interest in nitro fatty acids (NO2-FA) as signaling molecules formed under nitroxidative stress. NO2-FA were detected in vivo in a free form, although it is assumed that they may also be esterified to phospholipids (PL). Nevertheless, insufficient discussion about the nature, origin, or role of nitro phospholipids (NO2-PL) was reported up to now. The aim of this study was to develop a mass spectrometry (MS) based approach which allows identifying nitroalkenes derivatives of three major PL classes found in living systems: phosphatidylcholines (PCs), phosphatidylethanolamine (PEs), and phosphatidylserines (PSs). NO2PLs were generated by NO2BF4 in hydrophobic environment, mimicking biological systems. The NO2-PLs were then detected by electrospray ionization (ESI-MS) and ESI-MS coupled to hydrophilic interaction liquid chromatography (HILIC). Identified NO2-PLs were further analyzed by tandem MS in positive (as [M + H]+ ions for all PL classes) and negative-ion mode (as [M − H]− ions for PEs and PSs and [M + OAc]− ions for PCs). Typical MS/MS fragmentation pattern of all NO2-PL included a neutral loss of HNO2, product ions arising from the combined loss of polar headgroup and HNO2, [NO2-FA + H]+ and [NO2-FA − H]− product ions, and cleavages on the fatty acid backbone near the nitro group, allowing its localization within the FA akyl chain. Developed MS method was used to identify NO2-PL in cardiac mitochondria from a well-characterized animal model of type 1 diabetes mellitus. We identified nine NO2-PCs and one NO2-PE species. The physiological relevance of these findings is still unknown. itric oxide (•NO)-derived reactive nitrogen species (RNS) are potent oxidizing and nitrating agents, responsible for nitration and/or nitroxidation of biomolecules in vivo.1 In fact, nitrotyrosine is a reliable in vivo marker of nitroxidative stress conditions.2 RNS can react with unsaturated fatty acids to yield nitrated species, including the nitroalkenes derivatives of fatty acids or nitro-fatty acids (NO2-FA).3 The NO2-FA are endogenously occurring products of nitroxidative stress, which were detected in plasma,4−7 rat cardiomyocytes,8 human red blood cells,4 and other biological samples9 under physiological and pathophysiological conditions. Biological studies conducted on various NO2-FA indicated them to have an anti-inflammatory,10 antihypertensive,11 and antithrombotic properties.12 NO2-FA are usually detected in a negative ion mode mass spectrometry (MS), either by LC−ESI-MS approach or multiple reaction monitoring (MRM)-targeted analysis.4−7,10 Quantitative methods were also used in the

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detection of NO2-FA.4,13,14 Although most of the studies reported the presence of only endogenous NO2-FA species, these can also be found as protein adducts.15 Lipid nitration has been described to occurs in the hydrophobic environment of biological membranes.16 RNS easily diffuse, cross membranes, and concentrate into the hydrophobic milieu of the lipid membrane bilayers and lipoproteins.17−19 In consequence, the possibility of RNS to in vivo with esterified phospholipids fatty acid is evident, but this hypothesis has not been explored in previous studies. Thus, under nitroxidative stress conditions, formation of nitration products derived from the major PL species in cell membranes such as phosphatidylcholine (PC), Received: September 7, 2015 Accepted: January 26, 2016

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DOI: 10.1021/acs.analchem.5b03407 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry phosphatidylethanolamine (PE), and phosphatidylserine (PS), should be considered. The present study describes an in vitro model system to address PL nitration and to develop mass spectrometry based strategies to identify NO2-PL in biological samples. Lipid nitration was induced using nitronium tetrafluoroborate (NO2BF4). We have studied the fragmentation patterns of nitroalkenes derivatives of phospholipids, nitro phospholipids (NO2-PLs), in order to devise MS-based strategies for in vivo identification. The MS-based strategies were further validated by screening for NO2-PL species in cardiac mitochondria from a well-characterized animal model of type 1 diabetes mellitus (T1DM).

ions of each native phospholipid (nonmodified phospholipid). For details, see the Supporting Information. Characterization of NO2-PL in Cardiac Mitochondria. The animal care protocol, the induction, and characterization of STZ-induced hyperglycemia and the cardiac mitochondria isolation was performed as previously described.21 Lipid extraction of each mitochondrial fraction was performed according to the method of Bligh and Dyer.22 Total amount of PL was quantified with the phosphorus assay as previously described by Bartlett and Lewis.20 For details, see the Supporting Information. Hydrophilic Interaction Liquid Chromatography− Mass Spectrometry (HILIC-ESI-MS). Phospholipid from mitochondrial extracts from control and T1DM heart were separated by hydrophilic interaction liquid chromatography (HILIC-LC−MS) (Waters Alliance 2690) coupled online to a linear ion trap mass spectrometer (LXQ, ThermoFinnigan, San Jose, CA). Aliquots of 25 μg from the total lipid extract were diluted in 90 μL of the mobile phase B (acetonitrile 60%, methanol 40% with 10 mM ammonium acetate) and 10 μL of reaction mixture was introduced into an Ascentis Si HPLC Pore column (15 cm × 1.0 mm, 3 μm; Sigma-Aldrich). The solvent gradient was programmed as follows: gradient started with 0% of A (10% water, 55% acetonitrile and 35% methanol (v/v) with 10 mM ammonium acetate) and linear increase to 100% of A during 20 min and held isocratically for 30 min, returning to the initial conditions in 10 min. The LXQ linear ion trap mass spectrometer was operated in positive mode as described in the previous section. For details, see the Supporting Information. Theoretical Calculations. All calculations were performed with Gaussian0923 at the DFT level using the B3LYP functional and the 6-31++G** basis set. Wiberg bond indices were obtained from a Natural Bond Orbital 24 analysis as implemented in Gaussian software, at the same level of theory, using previously geometry optimized conformations. Statistical Analysis. For details, see the Supporting Information.

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EXPERIMENTAL SECTION Reagents/Chemicals. Phosphatidylcholine (PC 16:0/18:1, POPC; PC 16:0/18:2, PLPC; PC 16:0/20:4, PAPC; PC 14:0/ 14:0, dMPC), phosphatidylethanolamine (PE 16:0/18:1, POPE; PE 16:0/18:2, PLPE; PE 16:0/20:4, PAPE; PE 14:0/ 14:0, dMPE), and phosphatidylserine (PS 16:0/18:1, POPS; PS 16:0/18:2, PLPS; PS 16:0/20:4, PAPS) standards were purchased from Avanti Polar Lipids, Inc. (Alabaster) and used without further purification. Nitronium tetrafluoroborate (NO2BF4) was purchased from Sigma-Aldrich (Madrid, Spain). HPLC grade chloroform and methanol were purchased from Fisher Scientific Ltd. (Leicestershire, U.K.). Milli-Q water (Synergy, Millipore Corporation, Billerica, MA) was used and all reagents were used without further purification. Nitration of Phospholipids. Phospholipid nitration (125 μg of each PL) was carried out with nitronium tetrafluoroborate (NO2BF4; ≈1 mg) in chloroform (125 μL) at room temperature for 1 h, under orbital shaking at 750 rpm. Nitration was quenched with water (2 × 125 μL; vortexing for 30 s and centrifuged for 5 min at 4000 rpm), and the lower organic phase was dried under a nitrogen stream and stored at −20 °C to be further quantified using phosphorus assay20 and analyzed by ESI-MS and MS/MS. For details, see the Supporting Information. NO2BF4 has been previously used as a nitrating agent in fatty acid nitration studies and was chosen for mimicking the nitration occurring on biological membranes with peroxynitrite and nitrite.3−5,25 The concentration of NO2BF4 used was high allowing one to obtain nitrated derivatives of phospholipids in high amount to perform all the mass spectrometry structural characterization studies. Mass Spectrometry Conditions. The reactions were monitored by electrospray mass spectrometry in a linear ion trap mass spectrometer LXQ (ThermoFinnigan, San Jose, CA). An aliquot (4 μg) of each sample in methanol (2:100, v/v) was introduced through direct infusion. For the MS analysis of PCs in the negative-ion mode, 10 μL of 1 mM ammonium acetate in methanol were added and the samples were incubated for 5 min before the analysis. The LQX linear ion trap mass spectrometer was operated both in positive and negative ion modes. Data acquisition and analysis were performed using the Xcalibur Data System (version 2.0, ThermoFinnigan, San Jose, CA). Analysis of the NO2-PLs derivatives were also carried out by positive-ion mode ESI-MS and MS/MS, using a Q-TOF2 mass spectrometer (Micromass, Manchester, U.K.). Data acquisition and analysis were performed using the MassLynx 4.0 data system. The accurate mass measurements and elemental composition determination of the NO2-PL products were performed after locking the mass correction for the [M + H]+

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RESULTS AND DISCUSSION In this study, we have induced the nitration of PC, PE, and PS phospholipids, differing in degree of unsaturation of the hydrocarbon chains (oleic acid (OA), linoleic acid (LA), and arachidonic acid (AA)) (Table S-1 and Figure S-1), by incubation with nitronium tetrafluoroborate (NO2BF4). NO2BF4 is a nitronium cation salt (NO2+) that has been previously used as nitrating agent in fatty acid nitration studies and was chosen for mimicking the nitration occurring on biological membranes with peroxynitrite and nitrite,3−5,25 by allowing nitration to occur in hydrophobic environments.3,5,16 Tandem Mass Spectrometry Characterization of NO2PLs. Nitro-phospholipids, NO2-PLs, were observed in the positive mass spectra as [M + H]+ molecular ions and, in the negative mode mass spectra, as [M − H]− for PE and PS, and as [M + OAc]− molecular ions for PC. MS/MS spectra were acquired for both positive and negative precursor ions, to obtain complementary information, which could further be used for lipidomic studies on cell, tissues, and biofluids.26,27 In the mass spectra, the nitro-phospholipids (NO2-PLs) were observed with a mass increment of 45 u relative to the nonmodified PL (Table S-1). In order to confirm the formation of NO2-PLs, accurate mass measurement and elemental composition determination were performed in a Q-TOF mass B


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Analytical Chemistry

Figure 1. ESI-MS/MS spectra of (A) [M + NO2 + H]+ and [M + NO2 + OAc]− POPC molecular ions ([POPC + NO2 + H]+; A1, [POPC + NO2 + OAc]−); (B) [M + NO2 + H]+ and [M + NO2 − H]− POPE molecular ions ([POPE + NO2 + H]+; B1, [POPE + NO2 − H]−); and (C) POPS molecular ions ([POPS + NO2 + H]+; C1, [POPS + NO2 − H]−). Product ions indicating the presence of a NO2 group were labeled with (◆).

Figure 2. Major fragmentation pathways identified for NO2-PLs in positive- and negative-ion mode; OA-NO2-PLs derivatives were used as examples. Common fragmentations observed both in positive and negative modes are the loss of HNO2 and cleavages in the vicinity of the NO2 group. Fragmentations observed only in negative-ion mode ([M − H]− ions) corresponding to the carboxylate anions of NO2-FAs. Typical fragmentations observed in positive-ion mode ([M + H]+ ions) involve the presence of protonated NO2-FA and loss of polar headgroup typically observed for the cationized ions of PLs.

nitrated fatty acids as [NO2-FA + H]+ and [NO2-FA − H]−; (c) the fragmentation of the fatty acyl chain in the vicinity of the NO2 group; and (d) combined fragmentation of the NO2 group and the polar head groups of each PL. The fragmentation pathways identified for NO2-PLs are illustrated in Figure 2 using NO2-OA-PLs as a model. The product ions arising from observed fragmentation pathways can be used for identifying nitrated PLs. These include product ions formed due to neutral loss of the nitro group as HNO2 (loss of 47 u), observed both

spectrometer. On the basis of the accuracy of the measurements, the structural assignments could be made with a high degree of confidence (Table S-2). Further, MS/MS spectra were acquired for the PC, PE and PS nitro derivatives both in positive- and negative-ion mode (Figure 1) using collision induced dissociation (CID) conditions. All MS/MS spectra showed four main fragmentation pathways involving (a) the elimination of the nitro (NO2) group, as neutral loss of HNO2; (b) formation of ionized C


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of the [M + H]+ ions of NO2-PLs (Figure 1A−C) show fragmentations usually observed in the MS/MS spectra of cationized ions ([M + X, X = Na, K, Li]+) of nonmodified PLs.26,33−37 These fragmentations involve the polar headgroup and are the neutral losses of 59 u (C3H9N, trimethylamine) and 183 u (C5H14PO4N, phosphocholine) in the case of NO2-PCs. The decomposition of NO2-PEs and of NO2-PSs showed, respectively, the neutral loss of 43 u (aziridine, C2H5N) and the neutral loss of 87 u (Table S-3). The occurrence of these fragmentation pathways in NO2-PL MS/MS spectra can lead to misassignment of the peaks in lipidomics studies. This behavior can be caused by proton retention in the NO2 group, which modifies the lability of the chemical bonds, favoring different fragmentation pathways. Losses of 59 u, 43 u, and 87 u involves cleavages of the O−C bond, namely, between phosphate and the PL-headgroup. The presence of the charge in the NO2 group, rather than exclusively in the polar head, could favor the cleavage between the phosphate and the polar head, which only occurs in cationized non-nitrated PL. In fact, sodium ions have been reported to randomly associate with several regions and the free negatively charged phosphate oxygen can undergo fragmentation via neutral loss of trimethylamine (loss of 59 u).38 Bond Energy Theoretical Studies. In order to gain atomistic insights into the mechanisms governing the different fragmentation patterns of the several compounds, namely, the distinct fragments observed upon CID fragmentation of nitrated lipids, we have performed bond energy theoretical calculations. Since the distinct fragmentation was observed independently of the polar head and the fatty acyl chain, the theoretical calculations were performed considering the NO2PLPE system as a representative example of the common behavior of the remaining NO2-PLs studied. The PE was chosen since it has the simplest polar head in terms of chemical structure and is more prone to theoretical calculation. The strategy involved analysis of the Wiberg bond indices obtained through Natural Bonding Orbital (NBO) analysis at the B3LYP/6-31++G** level using geometry optimized molecules (at the same level and theory). To decrease the conformational freedom and the total number of atoms, the saturated aliphatic chain was truncated to a simple methyl substitute. The lowest energy conformations of the PLPE models protonated at the -NO2 and terminal -NH2 groups (top and bottom, respectively) alongside the calculated Wiberg bond indices of the relevant bonds are shown in Figure S-7. The lowest energy conformation for the -NO2 protonated model presents the phosphate OH hydrogen bonded to the ester carbonyl (dNH−O = 1.88 Å) whereas the ethanolamine terminal chain is stretched and not involved in any intramolecular interaction. In contrast, the -NH2 protonated structure shows an intramolecular hydrogen bond between one -NH3+ hydrogen and the ester carbonyl (dNH−O = 1.59 Å), resulting from the curling of the phosphoethanolamine fragment into itself. The protonation of the terminal NH2 originates a significantly more stable isomer, suggesting that this species are the most abundant. Comparing the Wiberg bond indices between both species, depicted in Figure S-7, the C−O bond near the ester is slightly weaker (lower bond index) in the NH2 protonated model (0.8674) than in the NO2 one (0.8768), suggesting that the former is more likely to be cleaved during MS/MS experiments. In contrast, the C−O bond close to the terminal NH2 is weaker in NO2 model than in the NH2 one, suggesting that cleavage of

in positive and negative mode MS/MS spectra of all NO2-PL, as can be seen in Figure 1 and Figures S-2 to S-4. Product ions arising from the neutral loss of H2O and 2H2O (loss of 18 u or 36 u) and combined loss of water and HNO2 (65 u and 83 u) were also observed. Such product ions were already reported for NO2-FA and thus can be used to unequivocally confirm the presence of a NO2 group.3−7,9,25,28−30 Other typical product ions that were observed in the MS/MS spectra are the protonated molecules of nitro-fatty acids, [NO2-FA + H]+, which occur due to cleavage between the fatty acid and the glycerol backbone, and which observation in MS2 is favored due to charge retention in the nitro group.31,32 These product ions were found at m/z 328.3 ([NO2-OA + H]+), m/z 326.3 ([NO2LA + H]+), and m/z 350.4 ([NO2-AA + H]+) (Figure 1A−C, Figure 2, and Table S-3). When NO2-PL were analyzed in the negative mode, we observed in all the MS/MS spectra peaks corresponding to the carboxylate anions of NO2-FA ([NO2-FA − H]−) (Table S-3). These ions allowed unequivocal identification of NO2-FA esterified to the particular PL. Furthermore, MS3 spectra of the nitro-fatty acids product ions, both in positive ([NO2-FA + H]+) and negative ion modes (NO2-FA − H]−), showed product ions confirming the presence of the NO2 group (neutral loss of HNO2), as reported previously for [NO2-FA − H]− (Figure S-5 and Table S3).8,16,28 As previously indicated, the third identified fragmentation pathway produces typical fragment ions arising from cleavage of fatty acyl chain in the vicinity of the NO2 group, as shown in Figure 2. This fragmentation pathway is observed in the MS/ MS spectra of both positive and negative NO2-PLs precursor ions. Formed product ions are common for the NO2-PLs with the same fatty acyl chain (OA, LA, and AA), independent of the polar headgroup. However, the fragment ions, that were observed for each fatty acyl chain were different and thus, the nature of the NO2-PLs was also different. In the case of NO2OA-PL, we have assigned the 9-NO2-OA-PL and 10-NO2-OAPL species, which is in accordance with previously reported results for oleic acid nitration.8,28 The location of the nitro group at C9 or C10 was inferred by the observed cleavage between C8−C9 or C10−C11 occurring with a neutral loss of 169 u (C10H19ON) or 128 u (C8H16O), respectively.8,28 For NO2-LA-PL, we have assigned three major isomers, with the nitro group attached at C9, C10, or C13, inferred by product ions formed by cleavages between C9−C10, C13−C14, C10− C11, and C12−C13, respectively. These backbone cleavages promote neutral losses of 156 u (C9H16O2), 86 u (C5H10O), 139 u (C8H13ON), and 115 u (C6H13ON), respectively. For NO2-AA-PLs, we assigned four major isomers, with the nitro group attached in C11, C12, C14, or C15. The location of the NO2 group at C14 and C15 were inferred by the observation of cleavages between C10−C11 and C13−C14 with the corresponding neutral loss of 183 u (C10H17O2N) or 143 u (C7H13O2N). The 11- or 12-NO2-AA-PLs were assigned due to neutral loss of 193 u (C12H19ON). Other minor product ions were observed, as reported in Figures S-5 and S-6, which are well described in the literature.8,28 These product ions can be used to locate the position of the NO2 group along the fatty acyl chain. Phospholipids usually show a typical MS/MS fragmentation pattern depending on the polar head, including the observation of a product ion at m/z 184 for [PCs + H]+ ions, and neutral losses of 141 u for [PEs + H]+ ions and of 185 u for [PSs + H]+ precursor quasi-molecular ions. However, the MS/MS spectra D


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nitro phospholipids in the animal model of type 1 diabetes mellitus (T1DM), using the knowledge of the typical fragmentation pattern of NO2-PL reported in the previous section. It is worth noting that, in the previous report, we did not identify any NO2-PL. In this study, samples were analyzed by HILIC-LC−MS and MS/MS. The retention time (RT) observed for PC class was between RT 45 and 57 min and for PE was between RT 22 and 30 min, based on elution of internal standards (dMPC and dMPE, respectively) (Figure S-12). The major PC molecular species identified for mitochondria of control and T1DM group, and their relative amount are shown in Figure 3A. The LC−MS/MS spectra were also interpreted using the fragmentation pathway of NO2-PCs data. By doing so, we were able to identify nine NO2-PC species in T1DM cardiac mitochondria at m/z 803.6 (NO2-PC 16:0/18:2); 805.7 (NO2PC 16:0/18:1); 851.6 (NO2-PC 18:2/20:4); 853.7 (NO2-PC 16:0/22:5, NO2-PC 18:0/20:5, and NO2-PC 18:1/20:4); 855.7 (NO2-PC 18:0/20:4); 857.6 (NO2-PC 18:2/20:1); and 879.6 (NO2-PC 18:0/22:6) (Figure 3B and Figure S-13). Only NO2PC 18:2/20:1 was detected in the control samples, although with a small relative abundance. As an example, the LC−MS/ MS spectra obtained for ions at m/z 805.7 (Figure 4) allowed one to confirm the presence of NO2-PC, based on typical fragmentation pattern, namely, loss of HNO2 and loss of HNO2+59 and HNO2 + 183. We calculated the ratio between the nitrated species and the corresponding nonmodified PC (Figure 3C). It was found that the ratio of NO2-PC/nonmodified PC was higher for PCs esterified with monounsaturated fatty acids such as for NO2-PC 34:1 (NO2-PC 16:0/18:1) at m/z 805.7, NO2-PC 38:3 (NO2PC 18:2/20:1) at m/z 857.6 and NO2-PC 38:5 (NO2-PC 18:1/ 20:4) at m/z 853.7. This is in agreement with a recent report showing that the increase in fatty acid length and in the unsaturation degree has a negative impact on the susceptibility toward NO2+ driven nitration.8 Using kinetic studies, authors reported that OA-NO2 was formed faster compared to nitrolinoleic, nitro-arachidonic, and nitro-docosahexaenoic acids. Interestingly, in our study, the nonmodified PC molecular species (PC 16:0/18:1 at m/z 760.6 (POPC), PC 18:2/20:1 at m/z 812.7 (GLPC), and PC 18:1/20:4 at m/z 808.6 (OAPC)) precursors of the NO2-PCs showing the higher percentage of conversion share the presence of one (mono)unsaturated fatty acyl chain in their composition. In this study, we were able to identify one nitrated PE species at m/z 837.5 (NO2-PE 18:0/22:6) formed from nitration of PE 18:0/22:6 at m/z 792.5 (SDPE) bearing docosahexaenoic acid (DHa) as unsaturated fatty acyl chain (Figure S-14). We have reported earlier the presence of PEs molecular species from cardiac mitochondria with high percentage of polyunsaturated fatty acids, mainly AA and DHa.21 This could justify the observed lack of nitrated PEs in T1DM cardiac mitochondria. Interestingly, rat primary cardiomyocytes treated with the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) showed increased levels of DHa-NO2 when compared with AA-NO2 (∼3-fold).8 The present results are significant as they highlight the presence of NO2-PCs and NO2-PEs derivatives in biological samples, subjected to oxidative stress, allowing the quantification of the percentage of conversion of nonmodified PL in NO2-PL. However, more research on this topic needs to be undertaken before the in vivo significance of phospholipid nitration is understood.

the latter is more probable. These results are in clear agreement with the fragmentation patterns observed experimentally. The MS/MS spectra of [NO2-PLs + H]+ ions (Figure 1A− C) also showed product ions arising from the combined loss of HNO2 and the polar headgroup. These were the product ions formed by the neutral loss of 59 + HNO2 and 183 + HNO2 for NO2-PCs, 43 + HNO2 and 141 + HNO2 for NO2-PEs, and 87 + HNO2 and 185 + HNO2 for NO2-PSs, as confirmed by MS3 analysis (Figures S-8−S-10). We also observed combined loss of HNO2 and neutral losses due to the cleavage in the vicinity of the NO2 group for [M + H]+ ions (Figures S-10 and S-11). Overall, the typical fragmentation pattern reported in here is crucial to design targeted approaches for the identification of NO2-PL in complex biological samples. Moreover, these can be used to propose specific multiple reaction monitoring (MRM), precursor ions scan (PIS), or neutral loss scan (NLS), essential to detect and quantify nitrated phospholipids in the lipidome, as is resumed in Table 1. Table 1. Tandem MS Fragment Ion for Diagnostic Scan of NO2-PLs in Positive and Negative Modes fragment ion/diagnostic scan positive mode [M + H]+ nitro group (NO2) Head Group phosphatidylcholine phosphatidylethanolamine phosphatidylserine Nitrated Modifications [OA-NO2]+ [LNO2]+ [AA-NO2]+ OA-NO2RCOO− LNO2RCOO− AA-NO2RCOO− HNO2 + Head Group phosphatidylcholine

phosphatidylethanolamine

phosphatidylserine

neutral loss of 47 (− HNO2) neutral neutral neutral neutral neutral neutral

loss loss loss loss loss loss

of of of of of of

59 u 183 43 141 87 185

negative mode [M − H]− neutral loss of 47 (− HNO2)

neutral loss of 87

product ion 328 product ion 326 product ion 350 product ion 326 product ion 324 product ion 348 neutral loss of 106 (HNO2 + 59u) neutral loss of 230 (HNO2 + 183u) neutral loss of 90 (HNO2 + 43u) neutral loss of 188 (HNO2 + 141u) neutral loss of 134 (HNO2 + 87u) neutral loss of 232 (HNO2 + 185u)

neutral loss of 134 (HNO2 + 87u)

Identification of NO2-PL in Mitochondria. Previously we have characterized the lipidome of cardiac mitochondria isolated from heart of an animal model of type 1 diabetes mellitus (T1DM).21 We observed an increase in the relative content of PC, and a decrease in the other classes of phospholipids (PI, SM, and PG) in T1DM mitochondria in comparison with control group. Additionally, we detected higher amounts of carbonylated and nitrated proteins in T1DM mitochondria in comparison with the control group.21 In consequence, we decided to further investigate the presence of E


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Figure 3. (A) Phosphatidylcholine (PC) molecular species profile observed for cardiac mitochondria of control group and type 1 diabetes mellitus (T1DM) group identified after LC−MS/MS analysis. (B) NO2-PC molecular species identified after LC−MS/MS analysis for cardiac mitochondria of control group and for type 1 diabetes mellitus (T1DM) group. The results were expressed as relative percentage obtained by dividing the ratio between the peak areas of each PC molecular species and the PC internal standard (dMPC) and the total of all ratio. Values are means ± standard deviation. *p < 0.05, **p < 0.01, and ***p < 0.0001, significantly different from the control group. (C) Ratio between nonmodified PC molecular species and NO2-PC in T1DM. The results were expressed as relative percentage determined by dividing the ratio between the peak areas of each NO2-PC molecular specie and the PC internal standard (dMPC) and the ratio between the peak areas of the correspondent nonmodified PC molecular species and the PC internal standard (dMPC). Values are means ± standard deviation. **p < 0.01 and ***p < 0.0001, significantly different from the control group.

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CONCLUSION

This approach allowed us to propose MS-based strategies that can allow simple, specific, rapid, and sensitive analytical methodologies for the in vitro and in vivo detection and characterization of phospholipid nitration products. These approaches include specific tandem mass spectrometry modes such as MRM, PIS, or NLS and can be used to identify and quantify NO2-PL in biological samples. On the basis of the typical fragmentation pattern observed for the NO2-PLs standards, we were able to identify 10 nitrated derivatives of phospholipids, 9 NO2-PCs, and 1 NO2-PE, in cardiac

In this study, we studied the in vitro formation of nitroalkenes derivatives of PCs, PEs and PSs after reaction with NO2BF4. Tandem mass spectra of the identified species (NO2-PC, NO2PE, and NO2-PS) were acquired in order to determine the main fragmentation pathways of NO2-PLs. The fragmentation pattern observed in the MS/MS spectra of all PLs classes showed product ions involving the neutral loss of HNO2, product ions formed by cleavage in the vicinity of the NO2 group and the presence of the nitrated fatty acyl chains. F



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mitochondria isolated from heart of a well-characterized animal model of type 1 diabetes mellitus. Nevertheless, more efforts are needed in order to test the proposed scan modes and to extend the identification of nitrated PLs in other conditions. This will contribute to the understanding of nitration mechanisms in biological environments and to establish the physiological relevance of NO2-PL species.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03407. Additional experimental details and results (PDF)

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REFERENCES

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Figure 4. Reconstructed ion chromatogram (RIC) and HILIC-ESIMS/MS spectrum of ions at m/z 805.7 ([M + H]+), retention time (RT) = 50.81 min, observed in mitochondria from T1DM heart.

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*E-mail: [email protected]. Phone: +351 234 370 698. Fax: +351 234 370 084. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to University of Aveiro, FCT/MEC, European Union, QREN, COMPETE for the financial support to the QOPNA Research Unit (FCT Grant UID/QUI/00062/2013) and UCIBIO Research Unit (Grant UID/Multi/04378/2013), through national funds and where applicable cofinanced by the FEDER, within the PT2020 Partnership Agreement, and also to the Portuguese Mass Spectrometry Network (Grant REDE/ 1504/REM/2005). Tânia Melo (Grant SFRH/BD/84691/ 2012) is grateful to FCT for her grant. Financial support from the European Regional Development Fund (ERDF, European Union and Free State Saxony, EFRE; Grants 100146238 and 100121468 to M.F.), COST Action CM1001, and stipend to I.M. provided by Universität Leipzig are gratefully acknowledged. G


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