Temporal Profiling of Peroxynitrite-Mediated Protein Nitration and Phosphorylation Using Proteomics Analysis

Peroxynitrite is capable of inducing protein nitration, which alters protein structure and interferes with signaling pathways dependent on phosphorylation. This study aims to investigate the effects of peroxynitrite-driven nitration and phosphorylation on proteins in HEK293T cells. Through label-free quantitative mass spectrometry, we successfully identified over 5,000 proteins from 0.5 μg of cell extracts collected at five different time intervals (0, 2, 15, 30, and 60 min) after exposure to peroxynitrite. We found that protein expression profiles from 2 to 60 min were distinct from those at 0 min. LC-MS/MS was also applied to quantify and map the nitration and phosphorylation sites. Analysis of protein nitration and phosphorylation revealed distinct temporal profiles, suggesting that nitration may contribute to the altered phosphorylation of the same protein or its interacting proteins. This study provides valuable insights for further mechanistic studies in peroxynitrite-related pathways.