Proteomic analysis of 6-nitrotryptophan-containing proteins in peroxynitrite-treated PC12 cells

.We identified 6-nitrotryptophan-containing proteins in peroxynitrite-treated PC12 cell, a neuron-like cell, by proteomic analysis using a specific polyclonal antibody which we have developed by using 6-nitrotryptophan-containing synthetic peptides as an antigen. Western blot analysis showed nine immunoreactive spots of newly appeared or significantly increased signals in the peroxynitrite-treated cells compared with that of the untreated cells in two-dimensional gel electrophoresis. We analyzed the proteins corresponding to these spots by trypsin digestion and LC-MS/MS. These spots contained seven proteins such as M2 pyruvate kinase, mitochondrial aconitase 2, eukaryotic translation elongation factor 2, pyruvate carboxylase, sarcosine dehydrogenase, vinculin and heat-shock protein 90α. Several proteins contained functional tryptophan residues that interact with other molecules. We speculate that the nitration of tryptophan residues in the neuron-like cell during neuroinflammation may affect cellular energy metabolism, protein synthesis, and stress response and may modulate the specific interaction of proteins with other molecules.