Identification of nitration sites by peroxynitrite on p16 protein

Injury of p16 has been implicated in some cancers. In this paper, we focus on the need for identification of peroxynitrite-dependent nitration sites on p16 with HPLC-MS/MS method. Two mono-nitrated residues Tyr129 and Tyr44 were detected in the course of p16 modification induced by peroxynitrite at relative low doses. As suggested by peptide mapping sequence analysis, Tyr44 was more liable to be nitrated by ONOO(-). Study on the chemical environment of two Tyr residues reveals that steric hindrance may be the structural determinant for the nitration sequence. Through technique of SDS-PAGE, ONOO(-) could induce p16 nitration, even strongly damage the combination of p16 with CDK4, which further influence p16's activity.