Quantification of 3-nitrotyrosine levels using a benchtop ion trap mass spectrometry method

Oxidative damage by reactive nitrogen species is linked to the pathogenesis of numerous inflammatory disorders, including atherosclerosis. 3-Nitrotyrosine (NO₂Tyr), a posttranslational modification of proteins generated by reactive nitrogen species, serves as a "molecular fingerprint" for protein modification by nitric oxide (NO)-derived oxidants. Studies demonstrate that systemic levels of protein-bound NO₂Tyr serve as an independent predictor of cardiovascular risks and are modulated by statin therapy. Measurement of NO₂Tyr in biological matrices may thus serve both as a quantitative index of nitrative stress in vivo and an important new prognostic marker of clinical relevance. Analytical methods for the accurate detection and quantification of trace levels of NO₂Tyr in biological tissues and fluids are, thus, of considerable interest. Here, we describe a rapid, sensitive, and specific method for the quantification of NO₂Tyr in biological matrices using readily available benchtop ion-trap mass spectrometry instrumentation (e.g., LCQDeca) combined with high-performance liquid chromatography (HPLC) interface. Through judicious use of stable isotopically labeled precursors as synthetic internal standards, the tandem mass spectrometric method described simultaneously adjusts for potential intrapreparative sample losses and monitors potential artifactual generation of NO₂Tyr during processing. The described method permits rapid and reproducible quantification of NO₂Tyr in biological and clinical specimens at the 100 fmol on column detection limit and should prove useful for studies defining the impact of reactive nitrogen species in cardiovascular disease and other inflammatory disorders.