Immunoaffinity liquid chromatography–tandem mass spectrometry detection of nitrotyrosine in biological fluids: Development of a clinically translatable biomarker

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC–MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022–2.20 nM, r2 = 0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.