Discovery of bioactive nitrated lipids and nitro-lipid-protein adducts using mass spectrometry-based approaches

Abstract

Nitro-fatty acids (NO₂-FA) undergo reversible Michael adduction reactions with cysteine and histidine residues leading to the post-translational modification (PTM) of proteins. This electrophilic character of NO₂-FA is strictly related to their biological roles. The NO₂-FA-induced PTM of signaling proteins can lead to modifications in protein structure, function, and subcellular localization. The nitro lipid-protein adducts trigger a series of downstream signaling events that culminates with anti-inflammatory, anti-hypertensive, and cytoprotective effects mediated by NO₂-FA. These lipoxidation adducts have been detected and characterized both in model systems and in biological samples by using mass spectrometry (MS)-based approaches. These MS approaches allow to unequivocally identify the adduct together with the targeted residue of modification. The identification of the modified proteins allows inferring on the possible impact of the NO₂-FA-induced modification. This review will focus on MS-based approaches as valuable tools to identify NO₂-FA-protein adducts and to unveil the biological effect of this lipoxidation adducts.

Highlights

• •Nitro-fatty acids (NO₂-FA) are endogenous bioactive lipids.
• •NO₂-FA form reversible Michael adducts with proteins leading to PTMs.
• •Adduction of NO₂-FA with proteins culminates to anti-inflammatory, anti-hypertensive, and cytoprotective effects.
• •Mass spectrometry (MS)-based approaches allows to identify NO₂-FA-protein adducts and to unveil their biological effects.

1. Introduction

During the last decade, nitrated lipid gained the interest of the scientific community, as new endogenous signaling molecules with important regulatory role in health and disease. Research on this is aimed at understanding the reactivity of reactive nitrogen species (RNS) with lipids, to unravel their occurrence in vivo and their biological roles. Among nitrated and nitroxidized lipids identified so far, the nitro-fatty acids (NO₂-FA) are best-known products of RNS. These products have been widely detected in several tissues [1], [2], [3], [4], [5] and biofluids [6], [7], [8], [9], [10], [11], [12], and are nowadays a hot topic in nitro lipidomics. NO₂-FA are considered important bioactive molecules and have been associated with anti-inflammatory [6], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], anti-hypertensive [25], [26], [27], [28], [29], [30], [31], [32], and anti-thrombotic properties [31], [33] and cytoprotective effects [2], [34], [35], [36], [37]. More recently, other nitrated and nitroxidized lipids [1], [6], [7], [8], [13], [38] and also nitro derivatives of phospholipids (PL) [39], [40] and triglycerides (TAG) [41] have been detected in biological samples and were associated with protective and beneficial effects, but they are scarcely studied. Also, esterified forms of NO₂-FA have been found as they can be generated either by direct nitration of the esterified fatty acyl moiety or by the incorporation of NO₂-FA [41].

NO₂-FA are also known as nitroalkenes derivatives of fatty acids since it includes a nitro group linked to the double bond (alkene group) of the unsaturated fatty acyl chain, and the nitro-alkene moiety makes these derivatives highly reactive with electrophilic properties. These endogenous electrophilic lipids are capable to covalently link to proteins, via Michael addition [42], leading to the formation of lipoxidation adducts. This type of post-translational modification (PTM) of proteins can modulate protein function, which underlies some of the biological roles attributed to the NO₂-FA (Fig. 1). Some of these PTMs are shown to elicit a protective effect, which may provide clues for new therapeutic strategies and new drugs.

Fig. 1.

Schematic representation of nitrated lipids pathways: from their generation to their biological effect.

Detection of NO₂-FA and especially their lipoxidation adducts are still a challenge that is mostly addressed by MS approaches. MS-based approaches have been extensively applied in the study of NO₂-FA-protein adducts [4], [7], [34], [42], [43], [44], providing detailed structural information of these adducts both in vitro and in vivo. LC-MS and MS/MS-based proteomics approaches have been performed to characterize the NO₂-FA protein adducts and the sites of adduction [34], [42], [45], [46], [47], [48]. Very recently peptide adducts were also reported for NO₂-FA esterified in phospholipids using biomimetic in vitro studies and MS approaches [49].

In this review we will discuss the formation and type of nitrated FA found in biological systems, their structure and reactivity with proteins and characterization by a MS-based proteomic and lipidomic approach that allowed to disclose possible biological roles associate with nitrated lipids-protein adducts.

2. Endogenous nitro-fatty acids

2.1. Chemistry and analysis

NO₂-FA are endogenous chemical entities generated by the attack of nitric oxide (NO)-derived reactive species, collectively called reactive nitrogen species (RNS), with unsaturated fatty acids. Nitrogen dioxide (^•NO₂), nitronium cation (NO₂⁺), and peroxynitrite/peroxynitrous acid, whose decomposition yields ^•NO₂ and hydroxyl radical (^•OH), were reported as RNS that most frequently initiate nitration or nitroxidation reactions in biomolecules, including lipids. The prevalence and the yield of one process of these processes over the others are dependent on the oxygen levels, concentration of ROS versus RNS, the presence of secondary target molecules (scavengers, thiols, and transition metals), pH, and the partition between hydrophilic and hydrophobic milieu in cellular compartments [50]. The mechanism of FA nitration and nitroxidation in biological systems is not yet wholly undisclosed, and there are some alternative routes to explain the generation of NO₂-FA (Fig. 2). The free radical-induced nitration of FA mediated by ^•NO₂ is one of the most prominent reaction in vivo as a source of NO₂-FA [51]. The endogenous formation of NO₂-FA during free radical-mediated nitration reactions occurs in several biological processes such as digestion [52], metabolic stress, and inflammatory conditions [53]. Thus NO₂-FA were already identified in human red blood cells [8], [9], plasma [6], [8], [9], [10], [12], urine [6], [7], and tissues [1], [2], [3], [4], [5] at concentrations ranging from picomolar [12] to micromolar [6]. Dietary sources of nitrite can also leads to the generation of NO₂-FA via acid-catalyzed nitration reactions [52], [54], [55], [56]. Recently, NO₂-FA were also reported in plants, fresh olives, and in extra virgin olive oil [56], [57], which are considered external sources of NO₂-FA and can contribute to rising the endogenous levels of NO₂-FA [58].

Fig. 2.

Representative mechanisms of nitro-fatty acid (NO₂-FA) formation. Radical-induced nitration of unsaturated fatty acids by nitrogen dioxide (^•NO₂) yields a β-nitroalkyl radical that can further react with other ^•NO₂ generating the nitronitrite intermediates. Further loss of nitrous acid (HNO₂) leads to the generation of the nitroalkene derivatives also called NO₂-FA. Electrophilic substitution at the double bond mediated by nitronium cation (NO₂⁺) also yields NO₂-FA.

The most common NO₂-FA identified in vivo were the nitrated forms of the nitro-oleic acid (NO₂-OA), nitro-linoleic acid (NO₂-LA), and nitro-conjugated linoleic acid (NO₂-cLA) [6], [8], [9]. However, the reaction of RNS with fatty acids can lead to the generation of several nitroalkene derivatives of other fatty acids, such as the nitro-palmitoleic acid (NO₂-POA), nitro linolenic acid (NO₂-LNA), nitro-arachidonic acid (NO₂-AA), nitro eicosapentaenoic acid (NO₂-EPA), and nitro-docosahexaenoic acid (NO₂-Dha) [6], [8], [38], [51]. Different stereo or positional isomers of NO₂-FA were detected in vitro and in biological samples [6], [8], [38], as represented in Fig. 3, Fig. 4, Fig. 5 and Table 1.

Fig. 3.

Proposed structures for nitro-oleic (NO₂-OA) and nitro-linoleic acids (NO₂-LA), with assignment of their different positional isomers, which were previously detected in in vitro studies and/or biological samples.

Fig. 4.

Proposed structures for nitro arachidonic acid (NO₂-AA), with assignment of its different positional isomers, which were previously detected in in vitro studies and/or biological samples.

Fig. 5.

Proposed structures for nitro-docosahexaenoic acid (NO₂-DHA), with assignment of its different positional isomers, which were previously detected in in vitro studies and/or biological samples.

Table 1.

Nitro-fatty acids identified in biological samples and in vitro mimetic model systems.

In vitromimetic model systems
NO2-FA	Isomer	Experimental model	Method	Ref.
Nitro-oleic acid (NO2-OA)
NO2-OA	9-NO2-OA	Gastric juice artificial + NO2−	C18-HPLC-ESI-MS and MS/MS in a API 4000 triple quadrupole and LTQ Orbitrap Velos	[56]
	10-NO2-OA
		Pancreatic lipase-digested EVOO
NO2-OA		MPO + H2O2 + NO2−	C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap (4000 Q-Trap)	[6]
		ONOO−
		NO2− in acidic conditions
NO2-OA	9-NO2-OA	●NO2	C18-HPLC-ESI-MS and MS/MS in API 2000 triple quadrupole	[80]
	10-NO2-OA
Nitro-linoleic acid (NO2-LA)
NO2-LA	9-NO2-LA	Gastric juice artificial + NO2−	C18-HPLC-ESI-MS and MS/MS in an API 4000 triple quadrupole and LTQ Orbitrap Velos	[56]
	10-NO2-LA
	12-NO2-LA	Pancreatic lipase-digested EVOO
	13-NO2-LA
NO2-LA		NO2− in acidic conditions	C18-HPLC-ESI-MS and MS/MS in a Quattro triple quadrupole	[10]
Nitro-conjugated linoleic acid (NO2-cLA)
NO2-cLA	8-NO2-cLA	Gastric juice artificial + NO2−	C18-HPLC-ESI-MS and MS/MS in an API 4000 triple quadrupole and LTQ Orbitrap Velos	[56]
	9-NO2-cLA
	11-NO2-cLA	Pancreatic lipase-digested EVOO
	12-NO2-cLA
NO2-cLA	9-NO2-cLA	MPO + H2O2 + NO2−	C18-HPLC-ESI-MS and MS/MS in an API 5000 triple quadrupole, API Q-Trap 4000, and Velos Orbitrap	[3]
		ONOO−
	12-NO2-cLA	●NO2
NO2-cLA	NO2-cLA	Photocontrollable peroxynitrite donor 2,3,5,6-tetramethyl-4- (methylnitrosoamino)phenol (P-NAP)	ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap (4000 Q-Trap)	[38]
	Multiple nitro, nitroso, and nitroxidized derivatives
Cholesteryl-nitro linoleic acid (Chol-NO2-LA)
Chol-NO2-LA		NO2− in acidic conditions	C18-HPLLC-ESI/MS/MS in a Quattro II triple quadrupole	[9]
Chol-NO2-LA		NO2− in acidic conditions	ESI–MS and MS/MS in a 2000 Q-Trap	[66]
			C18-HPLC-ESI–MS and MS/MS in a 2000 Q-Trap
Nitro-arachidonic acid (NO2-AA)
NO2-AA		●NO2	C18-HPLC-ESI-MS and MS/MS in an Esquire ion trap	[26]
NO2-AA	9-NO2-AA	NO2− in acidic conditions	C18-HPLC-ESI-MS and MS/MS in a hybrid quadrupole-linear ion trap	[51]
	12-NO2-AA
	14-NO2-AA
	15-NO2-AA
Biological samples
Nitro-palmitoleic acid (NO2-POA)
NO2-POA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitrohydroxy-palmitoleic acid (NO2OH-POA)
NO2OH-POA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitro-oleic acid (NO2-OA)
NO2-OA		Human red cells, plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
NO2-OA	9-NO2-OA	Myocardial heart tissue from a murine model of focal myocardial ischemia/reperfusion	C18-HPLC-ESI MS/MS	[1]
	10-NO2-OA
NO2-OA	NO2-OA and β-oxidation metabolites	NO2-OA acute intravenous treatment of mice with LPS-induced inflammation	C18-HPLC-ESI-MS/MS in an API 5000 triple quadrupole	[107]
NO2-OA	NO2-OA and its metabolic derivatives	Human and rat urine after intravenous administration of NO2-OA	C18-HPLC-ESI-MS and MS/MS in a LTQ Velos Orbitrap and API 5000 triple quadrupole	[61]
NO2-OA	NO2-OA and its metabolic derivatives	Mitochondrial extracts from rat hearts after ischemia-reperfusion	BME trans-nitroalkylation + C18-HPLC-ESI-MS and MS/MS in a 4000 Q trap hybrid triple quadrupole-linear ion trap	[62]
Dinitro-OA		Rat cardiomyocytes treated with peroxynitrite donor 3-	C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap (4000 Q-Trap)	[38]
		morpholinosydnonimine (SIN-1)
NO2-OA	NO2-OA and its Saturation, Desaturation	Plasma from NO2-OA-treated mice	C18-HPLC-ESI MS/MS coupled to an API 4000 hybrid triple quadrupole or API 5000 triple quadrupole	[59]
	β-oxidation metabolic derivatives
NO2-OA	NO2-OA saturation derivatives	NO2-OA-treated BAEC cells	C18-HPLC-ESI MS/MS coupled to an API 4000 hybrid triple quadrupole or API 5000 triple quadrupole	[59]
NO2-OA	NO2-OA and its derivatives	Liver lipid extracts from NO2-OA-treated mice	C18-HPLC-ESI MS/MS coupled to an API 4000 hybrid triple quadrupole or API 5000 triple quadrupole	[59]
Nitrohydroxy-oleic acid (NO2OH-OA)
NO2OH-OA		Human red cells, plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitro-linoleic acid (NO2-LA)
NO2-LA		Myocardial heart tissue from a murine model of focal myocardial ischemia/reperfusion	C18-HPLC-ESI MS and MS/MS	[1]
NO2-LA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
NO2-LA		Human blood plasma	C18-HPLC-ESI-MS and MS/MS in a Quattro triple quadrupole	[10]
NO2-LA	9-NO2-LA	Human red cell membranes and plasma	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap	[8]
	12-NO2-LA
NO2-LA	NO2-LA and its metabolic derivatives	Mitochondrial extracts from rat hearts after ischemia-reperfusion	BME trans-nitroalkylation + C18-HPLC-ESI-MS and MS/MS in a 4000 Q trap hybrid triple quadrupole-linear ion trap	[62]
Nitrohydroxy-linoleic acid (NO2OH-LA)
NO2OH-LA		Myocardial heart tissue from a murine model of focal myocardial ischemia/reperfusion	C18-HPLC-ESI MS and MS/MS	[1]
NO2OH-LA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitrokto-linoleic acid (NO2-oxo-LA)
NO2-oxo-LA		Myocardial heart tissue from a murine model of focal myocardial ischemia/reperfusion	C18-HPLC-ESI MS/MS	[1]
Nitro-conjugated linoleic acid (NO2-cLA)
NO2-cLA		Plasma and vaginal lavages after cLA inoculation in the vaginal lumen from mice infected intravaginally with HSV-2	C18-HPLC-MS/MS in a 6500+ Q-trap or a API 5000	[16]
Nitro-linolenic acid (NO2OH-LNA)
NO2-LNA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitrohydroxy-linolenic acid (NO2OH-LNA)
NO2OH-LNA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitro-arachidonic acid (NO2-AA)
NO2-AA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS into a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
NO2-AA		Rat cardiomyocytes treated with peroxynitrite donor 3-	C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap (4000 Q-Trap)	[38]
		morpholinosydnonimine (SIN-1)
Nitrohydroxy-arachidonic acid (NO2OH-AA)
NO2OH-AA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitro-Eicosapentaenoic acid (NO2-EPA)
NO2-EPA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitrohydroxy- Eicosapentaenoic acid (NO2OH-EPA)
NO2OH-EPA		Human plasma and urine	C18-HPLC-ESI-MS and MS/MS in a hybrid triple Q-linear ion trap (4000 Q-Trap)	[6]
Nitro-Docosahexaenoic acid (NO2-DHA)
NO2-DHA and dinitro-DHA		Rat cardiomyocytes treated with peroxynitrite donor 3-	C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap (4000 Q-Trap)	[38]
		morpholinosydnonimine (SIN-1)
Nitrohydroxy-Docosahexaenoic acid (NO2-DHA)
NO2OH-DHA		Rat cardiomyocytes treated with peroxynitrite donor 3-	C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap (4000 Q-Trap)	[38]
		morpholinosydnonimine (SIN-1)
Nitrohydroxy-Docosapentaenoic acid (NO2OH-DPA)
NO2OH-DPA		Rat cardiomyocytes treated with peroxynitrite donor 3-	C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap (4000 Q-Trap)	[38]
		Morpholinosydnonimine (SIN-1)
Nitro-conjugated linoleic acid (NO2-cLA)
NO2-cLA	9-NO2-cLA	Pancreatic lipase-digested EVOO	C18-HPLC-ESI-MS and MS/MS in an API 4000 triple quadrupole and LTQ Orbitrap Velos	[56]
	12-NO2-cLA
NO2-cLA	9-NO2-cLA	Urine of healthy humans	C18-HPLC-ESI-MS and MS/MS in a LTQ Velos Orbitrap and AB 5000 or API4000 Q-trap triple quadrupole	[7]
	12-NO2-cLA
	β-oxidation-metabolic derivatives of NO2-cLA
NO2-cLA	9-NO2-cLA	Rodents urine, plasma, and tissues (stomach, small intestine, colon, liver) after supplementation with cLA + NO2− and gastric acidification	C18-HPLC-ESI-MS and MS/MS in an API 5000 triple quadrupole, API Q-Trap 4000, and Velos Orbitrap	[3]
		Rodents liver and cardiac mitochondria incubated with NO2− in acidic conditions
	12-NO2-cLA
		Rodents cardiac tissue under ischemia-reperfusion
		Raw 264.7 macrophages stimulated with LPS/IFNγ
		Healthy human plasma
NO2-cLA	9-NO2-cLA	RAW264.7 macrophages stimulated with LPS/IFNγ and M1, M2 and M0 polarized bone marrow-derived macrophages (BMDM) treated with cLA	C18-HPLC-ESI-MS and MS/MS in an API 5000 or a Q-Trap 6500+ and LTQ Velos Orbitrap	[13]
	12-NO2-cLA
	Reduction and β-oxidation-metabolic derivatives
		Mice Peritoneal exudates after zymosan-A induced peritonitis
		and cLA supplementation
NO2-cLA	NO2-cLA and β-oxidation-metabolic derivatives	Urine and plasma healthy humans after ingestion of nitrite, nitrate and cLA	C18-HPLC-ESI-MS and MS/MS in a 5000 triple quadrupole	[58]
Cholesteryl-nitro linoleic acid (Chol-NO2-LA)
Chol-NO2-LA		Human blood plasma and lipoproteins from normolipidemic/healthy subjects	C18-HPLLC-ESI/MS/MS in a Quattro II triple quadrupole	[9]
Chol-NO2-LA		J774.1 macrophages timulated with LPS/IFNγ	C18-HPLC-ESI–MS and MS/MS in a 2000 Q-Trap	[66]

Nevertheless, the recovery of NO₂-FA from biological samples, together with their detection and accurate quantification is a challenge due to their low concentration, stability issues, metabolism (β-oxidation and saturation/desaturation reactions) [59], reactivity with proteins [59] and esterification [39], [40], [41], and different distribution among tissues and biofluids [6], [12]. In line with these limitations, there has been an effort for the development of specific, standardized and reproducible methodologies of sample preparation and sensitive analytical approaches. The advent of more sensitive and sophisticated instruments, allied with the possibility of high-throughput analysis prompted by MS-based approaches, combined or not with liquid chromatography (LC-MS), has been the selected tool for the identification, structural characterization and quantification of free NO₂-FA. Indeed, the detection of these lipids is an indication to disclose the bioactive properties of these nitrated derivatives. The progress in the field of MS-based approaches enabled the discovery of NO₂-FA and contributed to the knowledge of NO₂-FA biological roles giving information on the structure-function relationships [60]. The development of improved sample preparation techniques, chromatographic separations, high-resolution instruments with great sensitivity, and innovative tools raised the possibility of detection, structural characterization and quantification of nitro lipids in human samples and animal models both under physiological and pathological conditions [1], [3], [4], [8], [59], [61], [62], and also in plants [56] as summarized in Table 1. The identification of NO₂-FA by MS is based on the detection of specific mass shifts compared to non-modified fatty acid (FA+45 Da). Using MS-based approaches, NO₂-FA are preferentially analyzed in negative-ion mode as [M-H]⁻ ions [3], [63]. However, positive-ion mode ionization can also occur, and NO₂-FA can also be identified as [M+H]⁺ [26], [M+Li]⁺ [51], [M+NH₄]⁺ [9], [41], and [M+Na]⁺ ions [9]. Tandem mass spectra acquired both in positive- and negative-ion mode provides information that allows the structural characterization of NO₂-FA [8], [38], [51], [63], [64]. The fragmentation pattern of NO₂-FA under tandem MS (MS/MS) conditions includes the typical neutral losses of 47 Da (HNO₂) and product ions formed by cleavage of the hydrocarbon chain in the vicinity of the NO₂ group that allow assigning this modified FA. The differentiation of isomers can be addresses by the identification of reporter fragment ions that are formed by cyclization, followed by heterolytic carbon chain fragmentation, which allows pinpointing the correct position of the NO₂ group [2], [63]. These product ions have been used as diagnostic ions broadly employed for targeted analysis and quantitation of specific NO₂-FA using reversed phase LC-MS/MS approaches, in biomimetic systems and in cells, tissues and biofluids [3], [6], [8], [9], [10], [12], [17], [38], [59], [65], [66]. Structural information gathered by using MS studies can be further confirmed by infrared and nuclear magnetic resonance analysis for the confirmation of the functional groups and the final structure [6], [9], [10], [30], [31], [51].

The generation of NO₂-FA can be considered as the first step of nitration reactions. These species can be precursors of other nitrated and nitroxidized species because NO₂-FA can undergo additional reaction with ROS and RNS to be further nitrated, leading to the formation of nitroso, dinitroso, nitronitroso, di and trinitro species, or oxidized generating the assorted nitroxidized species as nitrohydroxy, nitrohydroperoxy, nitro-epoxy and nitro-keto (Table 1) [3], [6], [8], [26], [54], [64]. All of these derivatives were already identified by (LC)-MS and characterized by (LC)-MS/MS [1], [6], [9], [38]. In fact, the great sensitivity of MS-based approaches allowed to identify both nitro and nitrohydroxy derivatives of palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids in human plasma and urine [6]. However by far the NO₂-FA are the most studied mostly because, in opposition to other nitrated and nitroxidized FA, they are electrophilic molecules with great capability to react with protein with the formation of lipoxidation adducts.

2.2. Biological roles of nitro-fatty acids as new metabolic mediators, signaling molecules, and new therapeutic drugs candidates

NO₂-FA have raised the interest of the scientific community in last years, mainly because of their biological roles as key mediators in physiological and pathophysiological processes, as demonstrated in a variety of preclinical animal models of disease and in plants [2], [5], [13], [15], [20], [28], [32], [34], [45], [56]. They were assigned as biologically relevant and putative signaling molecules in cardiovascular disease [28], [33], myocardial ischemia/reperfusion and ischemia preconditioning [1], [2], [24], nephropathy [24], renal ischemia/reperfusion [24], diabetes and metabolic syndrome [14], pulmonary inflammation [15], [67] and chronic inflammatory disease [65]. NO₂-FA reach endogenous levels that allow them to mediate pivotal signaling actions as cytoprotective and pro-survival players [2], [34], [35], [36], [37], and based on their pleiotropic actions, NO₂-FA has emerged as potential therapeutic agents with high potential for therapeutic use (Table 2). In fact, NO₂-FA already undergo clinical trials [68]. The 10-NO₂-OA (CXA-10) demonstrated promising pharmacokinetic and pharmacodynamics characteristics during preclinical experiments [61], [68], [69]. CXA-10 is currently in phase II clinical trials for the treatment of chronic inflammatory and metabolic-related diseases, namely focal segmental glomerulosclerosis and pulmonary arterial hypertension, since it demonstrated beneficial effects when administrated via intravenous injections or through ingestion [61], [68], [69].

Table 2.

Modulation of target signaling pathways by NO₂-FA and related biological properties.

Anti-inflammatory
-NF-kB ↓ [1], [13], [15], [21], [23], [46], [60], [66], [86], [90], [108] -TL4R signaling ↓ [107] -PG H synthase ↓ [86], [109] -5-LOX ↓ [45] -STAT1 ↓ [87], [92] -Cytokine production ↓[1], [5], [7], [13], [20], [21], [23], [24], [66], [86], [110], [111], [112] -MPO ↓ [24] -Leukocyte recruitment, adhesion and infiltration ↓ [13], [23], [24], [87], [108], [112] -iNOS ↓ [13], [24], [36], [51], [60], [66], [86], [112] -COX-2 ↓ [112] -PGE2 ↓ [112] -Leukocytes number and activity ↓ [5], [13], [18], [108] -TNF-α ↓ [23] -Xanthine oxidoreductase (XOR) ↓ [73] -MKP-1 ↑ [92] -PPARγ ↑ [6], [15], [21], [65], [100] -TRPA-1 ↑ [98] -CD36 expression ↑ [100] -Heme oxygenase 1 (HO-1) ↑ [17], [19], [22], [23], [60], [66], [67], [89], [93] -STING ↓ [16] -PDI ↓ [95] -NADPH oxidase (NOX 2) ↓ [24], [74], [113]
Vasorelaxation
-Nitric oxide ↑ [29], [30], [31], [51] -eNOS ↑ [114] -Ang II-induced vasoconstriction ↓ [28]
Antioxidant
-Lipid peroxidation ↓ [71], [86] -Nrf2 ↑ [13], [15], [22], [46], [60], [88] -HO-1 ↑ [17], [19], [22], [60], [66], [89] -Reduction of protein nitration and oxidation [87] -Heat shock response (HSP) proteins [22] -XOR ↓ [73] -NOX 2 ↓ [24], [74], [113] -O2●-, ONOO- and NO production ↓ [36], [48], [86], [108], [111], [113]
Anti-hypertensive
-AT1R ↓ [28] -sEH ↓ [32] -Smooth muscle cell proliferation ↓ [111] -Prostaglandin F2↓ [111] -Differentiation of myofibroblast through Smad2 ↓ [113]
Anti-hyperglycemic
-PPARγ ↑ [6], [28], [100] -Glucose uptake ↑ [110] -Insulin sensitivity ↑ [6], [28], [100]
Anti-thrombotic
-Platelets activation and clotting ↓ [18], [31], [33] -Platelet production of thromboxane↓ [109] -Thrombin-induced aggregation ↓ [33] -PGHS ↓ [109], [115], [116]
Cytoprotective
-ANT1 ↑ [2], [34] -UCP-2 ↑ [2] -Mitochondrial dysfunction ↓ [36], [37] -Mitochondrial respiratory complex ↑ [48] -Metabolic shift ↑ [48]
Anti-tumorogenic
-Tumor cell proliferation, migration and invasion↓ [90]

The biological actions of NO₂-FA are mediated via a) decay reactions and transduction of nitric oxide (^•NO) signaling actions [29], [70], since NO₂FA can be considered NO donor; b) via receptor-dependent and c) via electrophilic adduction reactions to proteins [42], with formation of lipoxidation adducts. All these processes mediate important and specific signaling roles. These signaling actions are summarized in Table 2. Nitric oxide release by NO₂-FA has been associated with potential antioxidant properties through inhibition of lipid peroxidation process [71]. Additionally, the release of ^•NO by both NO₂-FA and nitrohydroxy FA derivatives has also been related with vasorelaxation properties of these nitrated lipid [26], [29], [30], [31], [51]. The nitro derivatives of arachidonic acid, NO₂-AA and nitrohydroxy-AA, were also reported to be able to release ^•NO and thus to induce cGMP-dependent vasorelaxation in rat aortic ring in an endothelium-independent manner [26], [31], [51]. NO₂-LA, NO₂-cLA and nitrohydroxy-LA promoted vessel relaxation via cGMP-dependent and endothelial-independent manner in pre-constricted rat aortic rings [29], [30]. Nevertheless, the ^•NO release by nitro lipids remains a controversial issue, and at some level, considered of minor relevance at endogenous levels [28], [29], [51], [70], [72]. Actually, ^•NO signaling actions mediated by NO₂-FA mainly occurs via cGMP-independent mechanisms. NO₂-FA modulates endothelial (eNOS) and inducible nitric oxide synthase (iNOS) gene expression and activity and consequently the eNOS- and iNOS-mediated ^•NO generation and reactions. Also, NO₂-FA modulates a broad array of signaling pathways that culminates with the downstream activation or inactivation of ^•NO signaling [67], [73], [74].

The covalent adduction to key proteins propelled by the electrophilic character of NO₂-FA seems to be the most prominent mechanism by which these nitro lipids spread their modulatory and protective actions. The identification and characterization of these NO₂-FA-protein adducts in distinct biological conditions have been achieved by reversed phase LC-MS-based proteomics approaches [28], [42], [56], [59], [75], [76]. This topic will be discussed in more detail in the next section.

As endogenous molecules, NO₂-FA undergo a series of metabolic, trafficking and clearance pathways that influences the regulation of activity, half-life and levels of free NO₂-FA. Protein adduction and esterification in complex lipids [70], [77], [78] are considered as reservoirs of NO₂-FA, allowing to regulate their endogenous levels [70], [77], [78]. NO₂-FA–protein adducts are reversible in biological systems [59], [73], [79] and NO₂-FA esterified forms can be hydrolyzed and mobilized by esterases and lipases, allowing NO₂-FA to return to free active forms [70], [80]. NO₂-FA can be metabolized via β-oxidation that mediates the formation of shorter and more polar electrophilic species [59] that retains the electrophilic power, but also to inactive nitroalkane species [7], [59]. In fact, in humans and rodents, the bio-elimination pathways of 10-NO₂-OA involves the generation of a series of shorter metabolites that were detected in urine using C18-HPLC-ESI-MS and MS/MS using both LTQ Velos Orbitrap and API 5000 triple quadrupole instruments [61]. However, the electrophilic functionality of NO₂-FA is irreversibly inactivated after reduction and conversion to the correspondent nitroalkane derivative by the nitroalkene saturase prostaglandin reductase-1 [81]. Both saturation and desaturation of the double bond of NO₂-FA are related with the generation of non-electrophilic NO₂-FA [59], which are nitro derivatives without signaling abilities. Adduction to peptides or proteins seems to have other proposes, such as the case of conjugation with GSH, which increases the urinary excretion rate of NO₂-FA excreted in urine [82]. Incorporation of NO₂-FA into lipoproteins is another way for NO₂-FA to enter in circulation and to be systemically distributed among tissues. The modulation of all of these diverse pathways will impact the potential reactivity, the efficacy of signaling actions and behavior of these nitration products.

The signaling actions of NO₂-FA are also mediated through the modulation of the structure and regulation of the expression and activity of anti- and pro-inflammatory proteins, heat shock proteins and phase II antioxidant response proteins. The capability of NO₂-FA to react with specific peptides and proteins determines the role of this nitrated lipids in redox regulation with consequence in cell signaling, as will be described in the following section.

3. Nitro-fatty acids and protein lipoxidation adducts

3.1. Main target and biological significance of PTM by nitro-fatty acids

NO₂-FA are electrophilic compounds, able to react via reversible Michael addition with nucleophiles within key proteins, leading to the formation of lipid-protein adducts (lipoxidation) in a process generally denominated nitroalkylation [83], [84]. The target nucleophiles in peptides and proteins include the deprotonated thiolate group of cysteine and the nucleophilic amino group of the imidazole moiety of histidine or the amino groups of lysine and arginine [83], [84], [85]. The high electronegative olefinic NO₂ group facilitate the addition to the double bond of the unsaturated hydrocarbon chain of NO₂-FA. This addition generates an important positive density of charge in the methylenic β-carbon adjacent to the nitration site. The oxygens of the NO₂ group withdraw electrons and the double bond is rearranged over the C–N bond, generating a carbocation. This conjugation makes the β-carbon adjacent to the NO₂ group electron poor and with potential reactivity. The NO₂-FA-protein covalent adducts generated during the nitroalkylation process are reversible, which seems to be related with the possibility of redox regulation [59], [73], [79] and thus can be associated with the apparent lack of toxicity of these modified lipids. All of the aforementioned characteristics make NO₂-FA as promising pharmacological compounds. In fact, pre-clinical and human trials has demonstrated the NO₂-FA favorable pharmacokinetics and safety.

The formation of NO₂-FA adducts with proteins is considered a key PTM of proteins. This modification of functionally-relevant proteins can modulate the patterns of gene expression programs, transcription factors function, enzyme function and activity, metabolic and inflammatory responses, and cell signaling networks [50], [59], [73], [84]. This lead to a series of downstream signaling events that are intrinsically related to the biological signaling roles of NO₂-FA [2], [6], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [61], [74], [75], [86], [87], [88], [89], [90] (Table 2). The activation of several of these pathways are considered essential for restoring the homeostasis and the redox balance and makes NO₂-FA promising pharmacological compounds [91].

There are several proteins reported to be targets of NO₂-FA electrophilic reactivity, for example, the p65 subunit of NF-κB [1], [23], [92], heme oxygenase-1 (HO-1) [17], [19], [22], [67], [89], [93], mitogen-activated protein kinase (MAPK) phosphatase 1 (MPK-1) [92], Kelch-like ECH-associated protein 1 (Keap 1) [17], [22], [46], [88], metalloproteinases (MMP-7 and MMP-9) [75], glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [42], [94], protein disulfide isomerase (PDI) [95], and transient receptor potential (TRP) channels [96], [97], [98], [99] (Table 3). NO₂-FA can also conduct their biological signaling roles by a receptor-dependent signaling action and peroxisome proliferator-activated receptor gamma (PPARγ) is one of the main targets, which is a significant route for the anti-inflammatory effect associated with NO₂-FA derivatives [6], [23], [47], [65], [93], [100], [101], [102].

Table 3.

Nitro-fatty acids lipoxidation adducts identified in biological samples and in vitro mimetic model systems by using mass spectrometry-based approaches.

Protein/peptide	Model system	Method	Molecular mechanism signaling action	Biological role	Ref.
Nitro-oleic acid (NO2-OA)
Cysteine	Incubation of NO2-OA and cysteine	C18-HPLC-MS and MS/MS in a triple quadrupole API 4000			[56]
	Incubation of NO2-OA and cysteine	C18-HPLC-MS and MS/MS in a triple quadrupole			[43]
	Whole olives, mesocarp and peel	C18-HPLC-MS and MS/MS in a triple quadrupole API 4000			[56]
GSH	NO2-OA-Cys adduct generation after incubation between NO2-OA and GSH	BME trans-nitroalkylation + C18-HPLC-ESI-MS and MS/MS in a Q trap 4000			[62]
	NO2-OA-Cys adduct generation after incubation between NO2-OA and GSH	(BME trans-nitroalkylation +) C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole API 4000 or API 5000 triple quadrupole			[59]
	Plasma from NO2-OA-treated mice	(BME trans-nitroalkylation) C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole API 4000 or API 5000 triple quadrupole			[59]
	NO2-OA-Cys adduct generation after incubation between NO2-OA and GSH	ESI-MS in LCQ ion trap			[42]
		Tryptic digestion + C18-HPLC-ESI-MS and MS/MS in an ESI-LCQ ion trap
		C18-HPLC-ESI-MS/MS in a Q-Trap 4000
	Red blood cells obtained from healthy humans	C18-HPLC-ESI-MS/MS in a Q-Trap 4000	Translocation to membrane	Regulation of enzyme function, cell signaling, and protein trafficking	[42]
GAPDH	Cytosolic and membrane-associated protein fractions from red blood cells obtained from healthy humans	SDS-PAGE under non-reducing and denaturing conditions + Tryptic digestion + C18-nanospray LC-MS and MS/MS in a LTQ ion trap	Translocation to membrane	Regulation of enzyme function, cell signaling, and protein trafficking	[42]
Cys149
His303
GAPDH	Incubation of NO2-OA and GAPDH	MALDI-TOF MS (Voyager DE PRO system)			[42]
Cys149
		C18-HPLC-MS in a LTQ ion trap
Cys153
		Tryptic digestion and MALDI-TOF MS (Voyager DE PRO system)
Cys244
His108
		Tryptic digestion + C18-HPLC-ESI-MS in an ESI-LCQ ion trap
His134
His327		Tryptic digestion + C18-nanospray LC-MS and MS/MS in a LTQ ion trap
GAPDH	Incubation of NO2-OA and GAPDH	Electrophoresis under reducing conditions + BME trans-nitroalkylation + C18-HPLC-ESI-MS and MS/MS in a 4000 Q trap hybrid triple quadrupole-linear ion trap			[62]
5-LOX	Incubation of NO2-OA and human recombinant 5-LOX	Tryptic digestion and C18-nanoHPLC-ESI-MS and MS in an Orbitrap XL	Irreversible inhibition of 5-LOX activity and	Anti-inflammatory	[45]
Cys416
Cys418
His125
His360	Incubation of NO2-OA and human polymorphonuclear leukocytes (PMNL) (intact and cell lysates)
			Prevention of lung injury and systemic immune responses
His362
His367
His372	NO2-OA treatment in murine model of LPS-induced inflammation (lung injury and cellular infiltration)
His432
Keap1	Incubation of NO2-OA and recombinant Keap1	Tryptic digestion C18-HPLC-MS and MS/MS in a LTQ	Release of Nrf2 tracsription factor to the nucleous for induction of expression of antioxidant phase II enzymes	Antioxidant	[46]
Cys38
Cys151
Cys226
Cys273	Human embryonic kidney (HEK)-293T cells transfected with recombinant Keap1 and treated with NO2-OA
Cys288
Cys257
Cys489
Catephsin S	Incubation of NO2-OA with a synthetic Cat S peptide (Cat S23–29)	C18-HPLC-MS and MS/MS in a Q Exactive Hybrid Quadrupole-Orbitrap	Downregulation of Cat S expression and activity	Tissue Protection	[5]
(Cat S)
				Anti-inflammatory
Cys25
Fp subunit of mitochondrial complex II	Incubation of NO2-OA with recombinant human complex II Fp subunit	Tryptic digestion and C18-HPLC-MS and MS/MS in a LTQ-XL	Inhibition of mitochondrial respiration complex II and O2●− production	Cytoprotective	[48]
His2
His5
His6
	Rat heart mitochondria treated with OA-NO2	Blue native electrophoresis, BME trans-nitroalkylation, C18-HPLC-MS amd MS/MS in a hybrid triple-quadrupole linear ion trap mass spectrometer (4000 Q trap)	Promotion of glycolysis	Antioxidant
Cys9
Cys14
AT1R	HEK293 cells overexpressing AT1R treated with NO2-OA	Immunoprecipitation of AT1R from cell lysates, BME trans-nitroalkylation reaction of AT1R-bound NO2-OA, and C18-HPLC-MS and MS/MS in a 4000 Q-Trap triple quadrupole	Inhibits AT1R-dependent vasoconstriction by reduction of heterotrimeric G-protein coupling and inhibition of IP3 and calcium mobilization	Anti-hypertensive	[28]
MMP-7	Incubation of NO2-OA with recombinant human proMMP-7 and proMMP-9	C18-HPLC-ESI-MS and MS/MS in a LTQ-XL	Modulation of proteolytic activity	Anti-inflammatory	[75]
Cys70
		C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap 4000 Q-Trap	Decrease of enzyme expression
MMP-9
Cys100
Albumin	Plasma from intraperitoneal NO2-OA-treated mice	Electrophoresis under reducing conditions + BME trans-nitroalkylation + C18-HPLC-ESI-MS and MS/MS in a 4000 Q trap hybrid triple quadrupole-linear ion trap			[62]
	Plasma from NO2-OA-treated mice	Electrophoresis + BME trans-nitroalkylation + C18-HPLC-ESI-MS and MS/MS in a hybrid triple quadrupole API 4000 or API 5000 triple quadrupole			[59]
PPARγ	Incubation of NO2-OA with human recombinant PPARγ LBD	Tryptic digestion and C18-HPLC-MS and MS/MS in a LTQ	Activation of PPARγ-related gene expression for glucose regulation and adipogenesis	Anti-hyperglycemic	[47]
	HEK 293 T cells were transfected with PPARγ and treated with NO2-OA	Immunoprecipitation, gel electrophoresis, BME- trans-nitroalkylation and ESI-MS and MS/MS in a hybrid triple quadrupole-linear ion trap mass spectrometer (4000 Q Trap,	Decrease in adipogenesis
Cys285			Increase glucose uptake	Anti-adipogenic effect
His266
His323			Restore insulin sensivity
His425
His449
STING	Incubation human STING-transfected HEK293T cells with 10-NO2-OA	Purification with magnetic beads, tryptic digestion and MALDI LTQ	Deregulation of STING palmitoylatio	Anti-inflammatory	[16]
Cys88
			Inhibition of STING signaling
Cys91		Orbitrap XL	Inhibition the release of type I IFN
His16
Nitro-linoleic acid (NO2-LA)
cysteine	NO2-LA-Cys adduct generation after incubation between NO2-LA and cysteine	(C18-HPLC)-ESI-MS and MS/MS in a triple quadrupole			[107]
GSH	NO2-LA-Cys adduct generation after incubation between NO2-LA and GSH	ESI-MS in LCQ ion trap			[42]
		C18-HPLC-ESI-MS/MS in a Q-Trap 4000
	NO2-LA-Cys adduct generation after incubation between NO2-LA and GSH	BME trans-nitroalkylation + C18-HPLC-ESI-MS and MS/MS in a Q trap 4000			[62]
	NO2-LA-Cys adduct	C18-HPLC/ESI/MS in Micromass Quattro II triple quadrupole			[76]
	Generation after incubation between NO2-LA and GSH
	Red blood cells obtained from healthy humans	C18-HPLC-ESI-MS/MS in Q-Trap 4000			[42]
	MCF7/WT and MCF7/MRP1–10 cells treated with NO2-LA	C18-HPLC/ESI/MS in Micromass Quattro II triple quadrupole			[76]
ANT1	NO2-LA-treated intact perfused hearts	Immunoprecipitation + SDS-PAGE + in-gel digestion tryptic digestion + ABSciex 5800 MALDI-TOF-TOF MS and MS/MS	Mitochondrial uncoupling	Cytoprotective	[34]
Cys57
Nitro-conjugated linoleic acid (NO2-cLA)
cysteine	NO2-cLA-Cys adduct generation after incubation between NO2-cLA and cysteine	C18-HPLC-MS and MS/MS in LTQ Velos Orbitrap and AB 5000 or API4000 Q-trap triple quadrupole			[7]
	Urine from healthy humans	C18-HPLC-MS and MS/MS in LTQ Velos Orbitrap and AB 5000 or API 4000 Q-trap triple quadrupole			[7]
	NO2-cLA-Cys adduct generation after incubation between NO2-cLA and cysteine	C18-HPLC-MS and MS/MS in API 5000 triple quadrupole			[44]
	Urine from healthy humans	C18-HPLC-MS and MS/MS in API 5000 triple quadrupole			[44]
Nitro-arachidonic acid (NO2-AA)
PDI	Incubation of human recombinat PDI with NO2-AA	C4-HPLC-MS of intact protein in a hybrid triple quadrupole/linear ion trap mass spectrometer (Q-trap 4500)	Inhibition of reductase and chaperone activity of PDI	Anti-inflammatory	[95]
		digestion tryptic digestion and C18-HPLC-MS and MS/MS
Cys397
Cys400

The nitro lipoxidation PTM of the proteins shown in the Table 3 have been correlated with specific biological effects. For example, nitroalkylation of the p65 subunit of NF-κB [23], induction of HO-1 expression [93], PPARγ modulation [100], inhibition of the correct assembly of the active NADPH oxidase (NOX2) [74], and inhibition of both reductase and chaperone activities of PDI and possible prevention of NOX2 activation [95] have been associated with the anti-inflammatory properties of NO₂-FA. Another important anti-inflammatory action of NO₂-FA is attributed to their capability to induce PTM of 5-Lipoxygenase (5-LOX) limiting the inflammation induced by the 5-LOX-dependent leukotriene synthesis. This point deserves to be further explored as a potential therapeutic/pharmacological strategy due to the physiological relevance of 5-LOX, namely in inflammation [45]. Induction of HO-1 and activation of Nrf2 have been correlated with protection against oxidative stress and antioxidant actions of NO₂-FA [93]. Activation of PPARγ by NO₂-FA has also been associated with glucose uptake and anti-hyperglycemic effects [100]. Inhibition of the catalytic activity of sHE was associated with anti-hypertensive properties of NO₂-FA [32]. Finally, neuroprotective effects associated with the decrease of protein aggregation were related with PTMs of α-synuclein by NO₂-OA [35].

3.2. Identification of protein-nitro-fatty acids adducts: tools and challenges

Identification of protein nitroalkylation by NO₂-FA has been disclosed by using different experimental approaches, as crystallographic analysis [100], [101], western immunoblot-based assay [2], [23], [32], [34], [87], spectrophotometry [7], [43], [44], [94] and MS-based approaches [4], [7], [34], [42], [43], [44]. However, spectrophotometry and immunoassays do not give detailed structural information and crystallography requires pure proteins, being difficult to be used in the analysis of complex biological samples.

Mass spectrometry, namely using matrix assisted laser desorption/ionization (MALDI) or electrospray (ESI) MS-based proteomics approaches, often coupled to reverse phase (RP) liquid chromatography (LC–MS), are the most suitable methods for detection and characterization of adducts formed between NO₂-FA and proteins. In vitro generation of NO₂-FA-protein adducts, in biomimetic systems, between standards of NO₂-FA and candidate peptides or proteins has been used as strategy for the initial identification by (LC)-MS and further characterization of these adducts by MS/MS. Data obtained using these biomimetic approaches using controlled reaction conditions are more straightforward and relatively easy to analyze. This, in turn, allows to obtain knowledge on the reactivity of each individual NO₂-FA and the typical fragmentation pathways under MS/MS needed to identify these adducts. The information gathered by tandem mass experiments concerning the typical fragmentation pathways and reporter ions can be used to identify these lipoxidation products in complex biological samples by using MS-based proteomics approaches and to develop MS target analysis, namely multiple reaction monitoring (MRM) analysis. This has contributed to achieve the ultimate goal that consists of the identification of the NO₂-FA-protein adducts in complex biological samples as cells, tissues, biological fluids, which requires specific and targeted approaches. Bottom-up proteomics approaches are usually performed. Through these analytical approaches, it is possible to unequivocally identify the modified peptides after enzymatic digestion of NO₂-FA-protein adduct, usually using trypsin, followed by the analysis of the tryptic peptides by reverse-phase (RP)-LC-MS and MS/MS. The addition of the NO₂-FA moiety increases the retention time of the modified peptides [42], which are identified on the mass spectra as singly, [M+H]⁺ ions, or multiple charged ions, [M+nH]ⁿ⁺, based on the mass shift against the unmodified peptide. This gives information on the nature of NO₂-FA covalently attached to the protein. The observed mass shift in the mass spectra for the Michael adducts will be equal to the molecular weight of the NO₂-FA. Thus, a mass shift of + 327 Da and + 325 Da corresponds to the addition of NO₂-OA and NO₂-LA, respectively [42]. MS/MS data allows to confirm the nature of the modification and provides information on the fragmentation pattern of NO₂-FA-peptide adducts. These data further allows to pinpoint the location of the modification site and thus the targeted residue in the peptide backbone [103], [104]. Detailed information to identify the sites of adduction is revealed by a mass shift of the typical b and y product ions of the adducted peptide, when compared with the non-modified one. The modified immonium ions are also useful to confirm the presence of a modified amino acid residue within the adducted peptide.

RP-LC-ESI-MS and MS/MS were used to detect lipoxidation adducts formed between NO₂-OA or NO₂-LA and GAPDH and GSH in vivo in healthy human red cells [42]. This methodology was also applied to confirm the post-translational modifications of matrix metalloproteinase by NO₂-OA [75], and for the identification of reversible Michael adducts of NO₂-OA and thiols of proteins and GSH in liver and plasma of NO₂-OA-treated mice [59]. Significant levels of protein cysteine adducts of NO₂-OA were also observed in fresh olives, especially in the peel [56]. AT₁-R adducts with NO₂-OA were quantified by HPLC-MS/MS using MRM scan mode in the negative-ion mode as BME adducts (BME-NO₂-OA adducts) after a nucleophilic exchange of NO₂-OA from AT₁-R to BME. The presence of exchangeable NO₂-OA demonstrated the direct adduction of AT₁-R by NO₂-OA, and therefore that AT₁-R is a relevant cellular target for NO₂-OA alkylation [28]. RP-LC-MRM scan in the positive-ion mode ([M+H]⁺ ions) was applied for the characterization of NO₂-LA-GSH adducts in vitro and further identification in MCF7 cells treated with NO₂-LA [76]. Nitroalkylation of albumin by NO₂-OA and NO₂-LA have been found in the plasma of mice gavage with these fatty acids [62]. Nitroalkylation of p65 subunit of nuclear factor κB (NF-κB) was observed in vivo in myocardial tissue of a murine model of ischemia-reperfusion with intravenous supplementation of OA and LA [1]. One study also reported the direct analysis by MALDI-TOF-TOF MS and MS/MS, in positive-ion mode, of adenine nucleotide translocase 1 (ATN 1) adducts after NO₂-LA infusion into intact perfused heats allowing to pinpoint that the nitroalkylation of ANT1 by NO₂-LA occurred on Cys57 [34]. Adduction of NO₂-OA to PPAR-γ [47], and to Keap1 [46] are also examples of biological detection and characterization of NO₂-OA-protein adducts by MS.

The Michael addition reactions between NO₂-FA and proteins is remarkably selective and depends on the nature and structural features of the NO₂-FA. The fatty acyl chain length and the position of the electrophilic carbon, i.e., the position of the nitroalkene group, has a pivotal effect on the reactivity of NO₂-FA [102]. Therefore both factors regulate the formation of NO₂-FA-protein adducts and the biological activity of the NO₂-FA [22], [42], [65], [73], [100], [101]. In spite of its four possible isomers (at C9, C10, C12 or C13), only the NO₂-LA isomers bearing the NO₂ at C10 and C12 were reported to selectively bind to cysteine 285 (Cys285) in the ligand-binding domain and activate PPARγ [101]. The C10 isomer of NO₂-OA is more reactive toward to Cys285 in the ligand binding domain of PPARγ than the C9 isomer [47]. On the other hand, Keap1 is easily activated by the C9 isomer via nitroalkylation of Cys273 and Cys288 [22], [46]. Xanthine oxidoreductase activity is preferentially inhibited by the C9 isomer of NO₂-OA or a mixture of both C9 and C10 isomers [73]. It has been reported that NO₂-FA with shorter acyl chains interact stronger with Nrf2 and NF-kB [60].

Overall, the identification of NO₂-FA-protein adducts is important, because it may give information, as shown in several examples reported earlier, on the potential protein targets whose modulation by NO₂-FA can have potential therapeutic interest.

4. Esterified nitro-fatty acids

4.1. Nitrated phospholipids and their lipoxidation adducts

In spite of their free forms, NO₂-FA can be stabilized by esterification in more complex lipids in hydrophobic compartments, as the biological membranes. Nitrated derivatives of phospholipids were identified in biomimetic model systems and also in vivo [39], [40]. In mimetic model studies, nitrated PLs were generated after in vitro incubation of PL standards (phosphatidylcholines, PCs and phosphatidylethanolamines, PEs) and NO₂BF₄, and its characterization was performed using C5-LC-MS and MS/MS in a Linear ion trap [39], [40]. Nitrated PCs and nitrated PEs were detected by HILIC-LC-MS and MS/MS-based lipidomic approaches in cardiac mitochondria from diabetic rats [39] and cardiomyoblasts subjected to starvation [40]. Nitrated 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (nitrated POPC) was reported to have antioxidant properties as scavenging agent, mediated by its anti-radical potential and ability to inhibit lipid peroxidation. Anti-inflammatory properties of nitrated POPC, related with its ability to inhibit iNOS expression in LPS-activated macrophages, were also reported [105].

NO₂-FA incorporation in PLs was also reported by using C18-HPLC-ESI-MS and MS/MS in API 4000 Q-trap triple quadrupole in adipocytes supplemented with NO₂-SA, NO₂-OA, NO₂-cLA, and NO₂-LA, before and after acidic hydrolysis. The incorporation yield and profile was specific for each supplemented NO₂-FA and PL class, being PC the PL class with highest levels of incorporation of NO₂-FA [106].

Nitrated POPC was also found to have the capability to form adducts with peptides. The identification of the covalent adducts of NO₂POPC with GSH was characterized by tandem MS in different instruments and the typical fragmentation pathways were disclosed for the first time. In this study, the NO₂POPC-GSH adducts were generated under biomimetic conditions and characterized by direct infusion MS and MS/MS using different instrumental platforms including LXQ linear ion trap, Q-TOF 2, and Q-Exactive Hybrid Orbitrap. The observed fragmentation pattern of NO₂POPC-GSH adducts included product ions that confirmed the presence of the phosphatidylcholine moiety (m/z 184.074 and neutral loss of 183 Da), the nitro group (neutral loss of HNO₂), and ^*y₂, ^*b₂ and ^*C₁ fragment ions of the modified peptide. All of these product ions pinpointing that NO₂POPC was linked to a cysteine residue of GSH (Fig. 6) and can be used as reporter ions applied in the search of these lipoxidation adducts in biological samples [49].

Fig. 6.

ESI-MS spectra of mono charged [M+H]⁺ (A) and double charged [M+ 2H]²⁺ ions (B) of NO₂POPC-GSH adducts, acquired in Q-Exactive Orbitrap, with identification of major fragmentation pathways. (Reprinted with permission from [49], copyright 2018 [John Wiley & Sons]).

4.2. Nitrated triacylglycerides

Nitrated triacylglycerides (NO₂-FA-TAG) have been reported in rat plasma after oral administration of NO₂-OA, together with β-oxidation and dehydrogenation derivatives of NO₂-FA-TAG in adipocytes supplemented with NO₂-OA. These studies were performed by C18-HPLC-ESI-MS and MS/MS in API4000 Q-trap triple quadrupole and LTQ Velos Orbitrap instruments [41]. Another study reported the differential esterification profile of NO₂-FA and their metabolites in TAGs in adipose tissue of rats fed with 10-NO₂-OA. By using C18-HPLC-ESI-MS and MS/MS in API 4000 Q-trap triple quadrupole, the NO₂-FA were observed to be preferentially incorporated in monoacyl- and diacylglycerides. This was found to be in opposite to its reduced metabolites, which were favorably incorporated in TAGs. These observations were corroborated by the analysis of the lipid polar and neutral fractions from adipocytes supplemented with NO₂-SA (nitro-stearic acid), NO₂-OA, NO₂-cLA, and NO₂-LA, after acidic hydrolysis [106].

The occurrence of nitrated phospholipids and triacylglycerides can be of high relevance at biological level. The NO₂-FA-containing phospholipids and triacylglycerides can act as a reservoir of NO₂-FA. Additionally, these esterified NO₂-FA can be further mobilized by lipases in turn to exert their adaptive and anti-inflammatory signaling actions. In the case of NO₂-FA-containing phospholipids, the NO₂-FA moiety seems to be able to retain the electrophilic character, and thus the ability to undergo reversible reactions via Michael addition with key proteins. Also, these phospholipid-esterified NO₂-FA can have an impact as anti-inflammatory and cytoprotective species. The nitration of esterified NO₂-FA or its incorporation into more complex lipids, together with the occurrence of lipoxidation products of NO₂-FA-containing phospholipids, and perhaps NO₂-FA-TAGs, can also contribute to the systemic distribution and metabolism of NO₂-FA.

5. Conclusion and future perspectives

NO₂-FA own important physiological functions that are mediated via formation of lipoxidation adducts and associated regulation of protein function. Several signaling proteins, with key roles in anti-inflammatory, anti-hypertensive, anti-hyperglycemic, and cytoprotective pathways, are targets of NO₂-FA adduction. This points to potential for new therapeutic strategies in important non-communicable diseases as cardiovascular, renal, pulmonary, and metabolic diseases. Mass spectrometry is a promising analytical tool in the detection of NO₂-FA-protein adducts. Nevertheless, there is a need for new methodological developments to improve the detection of these elusive lipoxidation adducts, and to obtain more insights regarding the protein targets of NO₂-FA and its roles in biological signaling pathways.