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 resu lting in peroxidation, oxidation, and/or nitration and as a consequenc e cause their inactivation. In this study mass spectrometry (MS) combi ned with both liquid (LC) and gas chromatography (GC) has been employe d to identify the products formed following ONOO- treatment of three p eptides at physiological pH: leucine-enkephalin (YGGFL), V3 loop (GPGR AF), and LVV-hemorphin7 (LVVYPW-TQRF), LC-MS analysis of leucine-enkep halin following ONOO treatment indicated the formation of products cor responding in mass to mono-and dinitrated forms of the starting materi al, LC-MS-MS and GC-MS analyses revealed no evidence for the formation of nitrophenylalanine; however, both 3-nitrotyrosine and 3,5-dinitrot yrosine 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 h ydrolysate. The main product was 3-nitrotyrosine which represented ca. 50% of the original tyrosine; traces of 3,5-dinitrotyrosine (ca. 3% o f the original tyrosine) were also present. (C) 1997 Academic Press.