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Characterization of Nitrophospholipid‐Peptide Covalent Adducts by Electrospray Tandem Mass Spectrometry: A First Screening Analysis Using Different Instrumental Platforms
Author(s) -
MonteroBullon JavierFernando,
Melo Tânia,
Domingues Maria Rosário,
Domingues Pedro
Publication year - 2018
Publication title -
european journal of lipid science and technology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.614
H-Index - 94
eISSN - 1438-9312
pISSN - 1438-7697
DOI - 10.1002/ejlt.201800101
Subject(s) - chemistry , popc , adduct , tandem mass spectrometry , peptide , electrospray ionization , mass spectrometry , electrospray , covalent bond , chromatography , biochemistry , phospholipid , organic chemistry , membrane
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 (NO 2 ‐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 (NO 2 ‐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 (NO 2 POPC), already detected in biological samples, and GSH peptide. The formation of NO 2 POPC‐GSH adducts is studied by ESI‐MS and MS 2 , 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 MS 2 conditions are modified b, y, and C ions bearing NO 2 POPC covalently linked that unequivocally confirms the presence of the lipid‐peptide adduct. Typical loss of HNO 2 is only observed in the MS 2 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 MS 2 allows the identification of the structure and specific MS 2 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.