Detection and quantitation of in vitro tyrosine nitration via selective reaction monitoring mass spectrometry

摘要

Nitrotyrosine formation is linear with respect to peroxynitrite concentrations between 5(mu)m and 100(mu)M. The characteristic UV absorbance of nitrotyrosine at 356 nm under acidic conditions is useful to confirm nitration (using either online or offline separation). Peroxynitrite-mediated nitration is selective. Although the mechanism driving selectivity is not yet understood, a SRM-based method should provide the specificity necessary to quantify site-specific nitration. Our workflow starting with nano-flow LC-high-resolution MS/MS followed by SRM can facilitate the identification of nitroproteins, determine and quantify site specificity of nitration, and allow for a sensitive and specific method that can be implemented for the site-specific quantitation of protein nitration in vivo. Synthetic stable-isotope labeled peptides will be used in future experiments to confirm nitroprotein identification and for absolute quantification of site-specific nitration (AQUA-basedstrategy).