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.
Yi
D
D
Biomedical Mass Spectrometry Unit, University of New South Wales, Sydney, Australia.
Smythe
G A
GA
Blount
B C
BC
Duncan
M W
MW
D002853:Chromatography, Liquid; D004743:Enkephalin, Leucine / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D015699:HIV Envelope Protein gp120 / Q000737:chemistry; D006454:Hemoglobins / Q000378:metabolism*; D013058:Mass Spectrometry; D009566:Nitrates / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D014297:Trimethylsilyl Compounds / Q000032:analysis / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D006454:Hemoglobins; D009566:Nitrates; D010446:Peptide Fragments; D014297:Trimethylsilyl Compounds; C107610:3,5-dinitrotyrosine; C039642:peroxynitric acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D004743:Enkephalin, Leucine; C047270:N,N-bis(trimethylsilyl)-2,2,2-trifluoroacetamide; C074309:LVV-hemorphin-7