Characterization of Nitrophospholipid-Peptide Covalent Adducts by Electrospray Tandem Mass Spectrometry: A First Screening Analysis Using Different Instrumental Platforms

Lipids are well-known targets of reactive nitrogen species and this reaction leads to the formation of nitrated lipids that have been associated with anti-inflammatory and cytoprotective effects. Nitro-fatty acids (NO2-FA) are highly electrophilic compounds that can form covalent adducts with proteins, leading to the formation of lipoxidation adducts,


Introduction
Phospholipids, main components of lipoproteins and cell membranes, are prone to be modified by reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to a plethora of oxidized or nitrated and nitroxidized lipids. Among these, oxidized lipids bearing terminal carbonyl group and nitrated lipids, are highly reactive electrophilic compounds, and can form covalent adducts with proteins, leading to the formation of lipoxidation adducts [1,2]. These reactions are main routes of protein post-translational modifications [3,4] being responsible for modulation of protein's structure and function. Most of the studies have been focused on lipoxidation adducts formed between peptides and proteins with electrophilic oxidized lipids [5,6] and, usually, these adducts are reported to have deleterious effects associated with inflammation and several diseases [1].
More recently, peptide and protein modifications by covalent adduction to nitrated fatty acids have been described [7] and have been associated with beneficial and health protective effects [7]. Nitro-fatty acids (NO 2 -FA) have been found both in vitro and in vivo, namely in red blood cells [8,9], plasma [8][9][10][11], and in different tissues, either in normal or in inflammatory conditions [12]. Several published works described the NO 2 -FA adducts with GSH [13,14], GAPDH [13,14], NF-κB [15] and PPARγ [16]. The type of post-translational modification seems to be an important regulatory pathway for the modulation of enzymatic activity, redox homeostasis and in signalling events [7,13,17] associated with inhibition of 4 inflammatory process and pro-survival responses. Also, NO 2 -FA and their protein adducts have been identified in olive and olive oil and were correlated with the beneficial effects of this food and of the Mediterranean diet [18].
The identification in vitro or in vivo of lipoxidation adducts and of nitrolipid-peptide/protein adducts, is usually done by using mass spectrometry (MS)-based approaches [13]. This approach allows to specify the electrophilic molecule that is covalently linked to peptide or protein, and to localize the addition site [5]. This structural information is obtained by analysis of the fragmentation pattern of peptide-electrophilic adducts obtained using tandem mass spectrometry experiments. [5,6].
Very recently, nitro-phospholipids (NO 2 -PL) were detected in vitro and in vivo systems, by our group [19,20], using MS-based approaches. Nitro derivatives of phosphatidylcholines (NO 2 -PC) and phosphatidylethanolamines (NO 2 -PE), mainly bearing oleic acid (OA) were detected in cardiomyocytes [20] and in cardiac mitochondria from diabetic rats [19]. These results showed that not only free NO 2 -FA, but also esterified NO 2 -FA in PL, can be a target of RNS and can be formed in vivo. NO 2 -PL was correlated with beneficial effects in the recovery phase in a cellular model of myocardium infarction in autophagy [20]. Also, NO 2 POPC showed antioxidant properties as scavenging agents and anti-inflammatory properties by inhibiting the expression of iNOS in Raw 264.7 macrophages stimulated with LPS [21].
However, there are no studies on the possible adduction of NO 2 -PLs to peptides. Thus, the aim of our work was to identify for the first time the typical tandem MS fragmentation pattern of the covalent adducts formed between GSH and NO 2 POPC, one of the NO 2 -PL species reported in biological samples [19,20], and that may have important biological properties. Analysis by tandem MS was performed in three different instruments commonly

Material and methods
Phospholipid nitration was carried out with nitronium tetrafluoroborate (NO 2 BF 4 ; Sigma-Aldrich, St Louis, MO, USA), as previously described [19,20,22]. A solution of 1-palmitoyl-2oleoyl-SN-glycero-3-phosphocholine (POPC; 1 mg; Avanti® Polar Lipids, Inc., Alabaster, USA) in chloroform (1 mL; Fisher Scientific Ltd., Leicestershire, UK) was prepared in an amber vial tube. Then, an excess of the solid NO 2 BF 4 (≈ 1 mg) was added. The vial containing the reaction mixture was purged with nitrogen stream, prior to the incubated at room temperature (20 °C) for 1 h, under orbital shaking at 750 rpm. This allow that the reaction occur under nitrogen atmosphere in order to avoid decomposition of the nitro phospholipid formed. After incubation, the reaction was stopped by solvent extraction with Milli-Q water.
The organic layer containing the phospholipid products was collected, and was dried under a nitrogen stream. The recovered nitrated PL was quantified using phosphorous assay and [NO 2 POCP+HF+Na] + is also observed at m/z 847.7, as observed previously for the nitration of fatty acids [22], and may correspond to the addition of fluoride to the double bond of the 6 monounsaturated fatty acid. Since nitroso or nitroxidized derivatives were not identified in our reaction, in contrast with what was reported before by Melo et al [20] and Milic et al [22] (because in the previously published work nitration was performed in the presence of air, favouring oxidation and decomposition of nitro POPC) and considering that the hydrofluoride nitro-POPC derivative lose the double bond, thus is not reactive toward protein/ peptides, the NO 2 POPC was used without further purification steps

Results and Discussion
The covalent adduct of NO 2  and are mainly accumulated at the membrane-water interface [23]. NO 2 -OA is able to shape the molecular organization of model membranes along the bilayer, and the nitro group is preferentially located near the head group of the phospholipid [23]. This arrangement facilitates the location of the nitro group more closely to the membranewater interface that favors the electrophilic adduction of the nitro-lipids with the anionic sulfhydryl moiety of the cysteine in peptides and proteins, in an aqueous medium.

Analysis of the MS/MS spectra of both mono and double charged ions, [M+H] + and
[M+2H] 2+ , allowed to identify the fragmentation pathways and to suggest reporter ions that can be further used to confirm the identity of these adducts. The MS/MS spectra obtained in three different mass spectrometers, an LXQ linear ion trap (CID), a Q-TOF (CID) and a Q Exactive Orbitrap (HCD) are shown in Figures 2 and 3. These MS platforms were selected because they are, nowadays, the most used for the detection of lipid -protein adducts either in biomimetic systems or in searching these compounds in biological samples analysis [13,24]. , and C 1 , now observed for the NO 2 POPC-GSH adduct, were also reported in tandem MS of the [M+H] + ions of the NO 2 -FA-GSH adduct [13]. In consequence, these product ions ( * y 2 , * b 2 , * C 1 ) can be considered as reporter ions for the target analysis of these nitro lipoxidation adducts in biological samples. However, in this case of the NO 2 FA adducts, loss of HNO 2 was not observed in the MS/MS spectrum but was only observed by MS 3 of * y 2 , when using the ion trap instrument [13].
The mass spectrometry approach used in this work was able to identify the formation of adducts between nitrated phospholipids and peptides, as exemplified for GSH. POPC is one of the most abundant phospholipids and it's nitration in vivo has been previously shown [19,20]. GSH is intracellularly biosynthesized but it is also found in the extracellular medium, as in plasma. In spite of being a hydrophilic peptide, it was chosen for this experiment due to its facility to oxidize and the availability of its cysteine residue. In fact, the susceptibility of being oxidized by the nitro-fatty acyl chain embedded in the bilayer of liposomes shows the accessibility for interaction between NO 2 -PL and peptides. This suggests the idea that a great number of peptides, but also proteins can be considered targets of nitrated phospholipids, especially those with a closer relationship with the membrane, which should be further investigated.

Conclusions
In summary, in this work we have characterized a nitro phospholipid-peptide adduct by MS and MS 2 . This approach allows to identify the nature of modified phospholipid that is covalently bound to the peptide/protein and the reported ions identified can be used to pinpoint these adductions in biological systems. The biological effects of these new kind of adducts remains to be studied but it can be considered as promising, based on the importance of nitroxidation events in physiological and pathophysiological circumstances.