Formation of nitrated tryptophan residues as a novel post-translational modification in the physiological state

Abstract

Protein nitration is a post-translational modification that is induced by reactive nitrogen species (RNS) such as peroxynitrite (ONOO−) and nitrogen dioxide (NO2). Nitration of tyrosine residues in proteins has been studied extensively and found in many diseases under oxidative stress. We have found a novel type of protein nitration, formation of 6-nitrotryptophan (6-NO2Trp) residues, developed its specific antibody, and constructed a method to detect 6-NO2Trp-containing proteins by proteomic analysis using LC–ESI–MS–MS (1). In this study, we applied this method for PC12 cell (naïve PC12 cell) and NGF-treated PC12 cell (differentiated-PC12 cell) to detect 6-NO2Trp-containing proteins in physiological state. We found several peptides from five ribosomal proteins (RP) (60S RP L7, 60S acidic RP P1, 40s RP S2, 40S RP S6, and 40S RP S19), 14-3-3 protein epsilon, nucleoside diphosphate kinase B, and proteasome subunit alpha type1 as nitrotryptophan containing peptides. We successfully determined the positions of nitrated tryptophan residues in the amino acid sequences of these proteins. Among them, the tryptophan nitration was observed only in the differentiated-PC12 cells for 40S RP S19 protein and 14-3-3 protein. The nitrotryptophan positive signals of 40S RP S19 and 14-3-3 protein were suppressed by treatment of PC12 cells with NOS inhibitor, L-NAME. The tryptophan nitration was not observed by LC–MS–MS analysis of these proteins. Finally, we also found several tryptophan-nitrated proteins in brain of adult rats by using the same method. Reference [1] H. Kawasaki et al., Free Radic. Biol. Med. 50 (2011) 419–427.