Peroxynitrite-Mediated Nitration of Peptides: Characterization of the Products by Electrospray and Combined Gas Chromatography–Mass Spectrometry

Peroxynitrite (ONOO−) can react with a wide range of biomolecules resulting in peroxidation, oxidation, and/or nitration and as a consequence cause their inactivation. In this study mass spectrometry (MS) combined with both liquid (LC) and gas chromatography (GC) has been employed to identify the products formed following ONOO−treatment of three peptides at physiological pH: leucine–enkephalin (YGGFL), V3 loop (GPGRAF), and LVV-hemorphin7 (LVVYPWTQRF). LC–MS analysis of leucine–enkephalin following ONOO−treatment indicated the formation of products corresponding in mass to mono- and dinitrated forms of the starting material. LC–MS–MS and GC–MS analyses revealed no evidence for the formation of nitrophenylalanine; however, both 3-nitrotyrosine and 3,5-dinitrotyrosine were observed and characterized. GC–MS analysis of hydrolyzed peptides following ONOO−treatment confirmed the presence of nitrated and dinitrated tyrosine. However, when a 20-fold molar excess of ONOO−was reacted with leucine–enkephalin, only about half of the tyrosine originally present in the peptide could be accounted for in the acid hydrolysate. The main product was 3-nitrotyrosine which represented ca. 50% of the original tyrosine; traces of 3,5-dinitrotyrosine (ca. 3% of the original tyrosine) were also present.