Recent methodological advances in the mass spectrometric analysis of free and protein-associated 3-nitrotyrosine in human plasma

l-Tyrosine and l-tyrosine residues in proteins are attacked by various reactive-nitrogen species (RNS) including peroxynitrite to form 3-nitrotyrosine (NO2Tyr) and protein-associated 3-nitrotyrosine (NO2TyrProt). Circulating NO2Tyr and NO2TyrProt have been suggested and are widely used as biomarkers of oxidative stress in humans. In this article the mass spectrometry (MS)-based analytical methods recently reported for the quantification of circulating levels of NO2Tyr and NO2TyrProt are discussed. These methodologies differ in sensitivity, selectivity, specificity and accessibility to interferences with the latter mainly arising from artifactual formation of NO2Tyr and NO2TyrProt during sample treatment such as acidification and chemical derivatization. Application of these methodologies to healthy normal humans revealed basal circulating levels for NO2Tyr which range between 0.7 and 64 nM, i.e. by two orders of magnitude. Application of gas chromatography-tandem mass spectrometry (GC-tandem MS) methods by two independent research groups by using two different protocols to avoid artifactual nitration of l-tyrosine revealed almost identical mean plasma levels of the order of 1.0 nM in healthy humans. The lower limits of quantitation (LOQ) of these methods were 0.125 and 0.3 nM, respectively. This order of magnitude for basal NO2Tyr is supported by two liquid chromatography-tandem mass spectrometry (LC-tandem MS) methods with LOQ values of 4.4 and 1.4 nM. On the basis of the data provided by GC-tandem MS and LC-tandem MS the use of a range of 0.5-3 nM for NO2Tyr and of 0.6 pmol/mg plasma protein or a molar ratio of 3-nitrotyrosine to tyrosine in plasma proteins of the order of 1:106 for NO2TyrProt in plasma of healthy humans as reference values appear reasonably justified. Recently reported clinical studies involving 3-nitrotyrosine as a biomarker of oxidative stress are discussed in particular from the analytical point of view.