A qualitative and quantitative study of nitrotyrosine using mass spectrometry and immunochemistry

Abstract: Background: Reactive oxygen (ROS), and nitrogen (RNS) species are formed during normal metabolism and in higher concentration in pathological conditions. They may cause cellular damage when the antioxidant defence is unable to balance the production of oxidants. Oxidative stress may cause a direct modification of amino acid side chains, such as the nitration of the tyrosine to nitrotyrosine. Its identification has relied on antibody assays or quantitative methods, but the results are contradictory.Aim: In this work, we have assumed that inflammatory-associated diseases involve RNS and we have hypothesised that nitrotyrosine may be a key marker of NO-derived reactive species. Our objectives were to develop methods capable of quantitatively and qualitatively identifying circulating and protein-incorporated nitrated tyrosine residues. Metods: Mass spectrometry (MS) has been used in connection with different ionisation techniques in order to identify both the actual nitration site in proteins and the circulating amount in the body fluids. Due to low levels and the intrinsic nature of amino acids, derivatisation of nitrotyrosine was necessary to create a sensitive and specific quantitative method. We made great efforts to rule out false nitration as a component of the quantified amount of nitrotyrosine. Results: By employing electrospray (ES) and tandem MS, we were able to identify 25% of the nitrated tyrosine sites of in vitro nitrated serum albumin. By investigating nitrated proteins using immunochemistry and immunohistochemistry, we found that the expressed antigen-antibody reactions were antibody, temperature and heme-dependent, thereby indicating the importance of further methodological considerations. By using GC-tandem MS analysis of the free form of nitrotyrosine in a rat inflammation model, we found that circulating nitrotyrosine accumulated in plasma during the experimental period, while the nitric oxide metabolite, nitrate, fluctuated in a biphasic manner. In normal human plasma, our new derivatization assay yielded basal nitrotyrosine levels among the lowest reported so far, probably because our method does not incorporate false nitration. Conclusion: According to our mass-spectrometric method, free circulating nitrotyrosine levels may be lower than those previously suggested. The nitration of proteins appears to be selective and our findings indicate that the nitration of proteins with subsequent degradation is a major source of circulating free nitrotyrosine.