AbstractProtein tyrosine nitration is a post‐translational modification commonly used as a marker of cellular oxidative stress associated with numerous pathophysiological conditions. We focused on ubiquitin carboxyl terminal hydrolase‐L1 (UCH‐L1) and glyceraldehyde‐3‐phosphate (GAPDH) which are high‐abundant brain proteins that have been identified to be highly susceptible to oxidative modification. Both UCH‐L1 and GAPDH have been linked to the pathogenesis of Alzheimer's and Parkinson's disease, however specific nitration sites have not been elucidated. Identification of specific nitration sites and quantitation of endogenous nitrated proteins are important in correlating this modification to disease pathology. In this study, purified UCH‐L1 and GAPDH were nitrated in vitro with peroxynitrite and the presence of nitrated proteins was confirmed by anti‐3‐nitrotyrosine Western blots. Data‐dependent LC‐MS/MS analysis identified several distinct tyrosine nitration sites in UCH‐L1 (Tyr‐80) and GAPDH (Tyr‐47, Tyr‐92, and Tyr‐312). Subsequent validation with synthetic peptides was conducted for selected nitropeptides. An LC‐MS/MS method was developed for semi‐quantitative determination of the synthetic nitropeptides: KGQEVSPKVY*(UCH‐L1) and mFQY*DSTHGKF (GAPDH). The nitropeptides were detectable in the mid‐attomole range and the peak area response was linear over three orders of magnitude. Targeted analysis of endogenous UCH‐L1 and GAPDH nitration was then conducted in an in vivo second‐hand smoke rat model to evaluate the utility of this approach.