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

Abstract

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, which modulate the protein structure and function. Nitrated phospholipids (NO2‐PL) have been detected recently in biological samples, but their biological effects are unknown, although similarly to what has been described for nitrated lipids, it has been hypothesized that they may react with peptides and proteins. In this study, in vitro biomimetic assays are used to synthetize adducts of nitrated POPC (NO2POPC), already detected in biological samples, and GSH peptide. The formation of NO2POPC‐GSH adducts is studied by ESI‐MS and MS2, using both low and high energy CID in different MS platforms: a LXQ linear ion trap, a Q‐TOF 2, and a Q‐Exactive Hybrid Quadrupole‐Orbitrap. Typical product ions observed under MS2 conditions are modified b, y, and C ions bearing NO2POPC covalently linked that unequivocally confirms the presence of the lipid‐peptide adduct. Typical loss of HNO2 is only observed in the MS2 of the mono‐charged precursor ions, [M+H]+. Product ions at m/z 184 or neutral loss of 183 Da are assigned as typical fragmentations that confirm the presence of the phosphatidylcholine. In summary, the characterization of nitro PL‐peptide adducts by MS and MS2 allows the identification of the structure and specific MS2 reporter ions to be used to pinpoint these adductions in biological systems. Practical Applications: The covalent interaction between nitro phospholipids and peptides suggests a new pathway in cellular transduction of nitroxidative stress signal. This adduction can be considered a post‐translational modification (PTM) of lipoxidation type, similarly as it has been described for nitro‐fatty acids and with important physiological implications. The identification and characterization of the nitro phospholipids and peptides adducts are possible by MS and tandem MS. This analytical technique also represents a robust and sensitive approach for detection of nitro‐lipids adduction to peptides or proteins in biological samples, allowing to disclose their physiological and clinical implications. Tandem MS fingerprinting is an essential feature for this purpose, so in this work, the identification of the reporter ions typical for this type of lipoxidation adducts is provided. These reporter ions can be used to design target (NL, PIS, or MRM) approaches to detect these type of PTM in biological environments. Nitrated phospholipids can adduct covalently to nucleophilic peptide gluthathione, as demonstrated by characterization using tandem MS.

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

“…All of these product ions pinpointing that NO 2 POPC 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. 6ESI-MS spectra of mono charged [M+H] + (A) and double charged [M+ 2H] 2+ ions (B) of NO 2 POPC-GSH adducts, acquired in Q-Exactive Orbitrap, with identification of major fragmentation pathways.

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“…6ESI-MS spectra of mono charged [M+H] + (A) and double charged [M+ 2H] 2+ ions (B) of NO 2 POPC-GSH adducts, acquired in Q-Exactive Orbitrap, with identification of major fragmentation pathways. (Reprinted with permission from [49], copyright 2018 [John Wiley & Sons]).…”

“…LC-MS and MS/MS-based proteomics approaches have been performed to characterize the NO 2 -FA protein adducts and the sites of adduction [34], [42], [45], [46], [47], [48]. Very recently peptide adducts were also reported for NO 2 -FA esterified in phospholipids using biomimetic in vitro studies and MS approaches [49].…”

Understanding the nitrolipidome: From chemistry to mass spectrometry and biological significance of modified complex lipids

“…The NO 2 derivatives of PC (16:0/18:1), NO 2 -POPC, have also been detected in human adrenal cortex adenocarcinoma cells (SW13/cl.2 cells) treated with 10 µM nitrated POPC [48,63] (Figure 15). In this case, the presence of modified species may occur due to their uptake to become part of the cell membrane or may suggest an increase in nitroxidative stress which leads to the generation of nitrated and nitroxidized species from the PLs that incorporate the membrane.…”

Insight into the cellular effects of nitrated phospholipids: Evidence for pleiotropic mechanisms of action

“…Additionally, their spontaneous decay could give rise to other reactive species [1]. Recently, the formation of adducts of NO 2 -POPC with glutathione (GSH) has been shown by mass spectrometry (MS) approaches [65], although the extent of formation is low. To the best of our knowledge, NO 2 -PL adducts with proteins have not yet been detected.…”