Mass spectrometry and 3-nitrotyrosine: Strategies, controversies, and our current perspective

Abstract

Reactive-nitrogen species (RNS) such as peroxynitrite (ONOO(-)), that is, the reaction product of nitric oxide ((•)NO) and superoxide (O2(-•)), nitryl chloride (NO2Cl) and (•)NO2 react with the activated aromatic ring of tyrosine to form 3-nitrotyrosine. This modification, which has been known for more than a century, occurs to both the free form of the amino acid (i.e., soluble/free tyrosine) and to tyrosine residues covalently bound within the backbone of peptides and proteins. Nitration of tyrosine is thought to be of biological significance and has been linked to health and disease, but determining its role has proved challenging. Several key questions have been the focus of much of the research activity: (a) to what extent is free/soluble tyrosine nitrated in biological tissues and fluids, and (b) are there specific site(s) of nitration within peptides/proteins and to what extent (i.e., stoichiometry) does this modification occur? These issues have been addressed in a wide range of sample types (e.g., blood, urine, CSF, exhaled breath condensate and various tissues) and a diverse array of physiological/pathophysiological scenarios. The accurate determination of nitrated tyrosine is, however, a stumbling block. Despite extensive study, the extent to which nitration occurs in vivo, the specificity of the nitration reaction, and its importance in health and disease, remain unclear. In this review, we highlight the analytical challenges and discuss the approaches adopted to address them. Mass spectrometry, in combination with either gas chromatography (GC-MS, GC-MS/MS) or liquid chromatography (LC-MS/MS), has played the central role in the analysis of 3-nitrotyrosine and tyrosine-nitrated biological macromolecules. We discuss its unique attributes and highlight the role of stable-isotope labeled 3-nitrotyrosine analogs in both accurate quantification, and in helping to define the biological relevance of tyrosine nitration. We show that the application of sophisticated mass spectrometric techniques is advantageous if not essential, but that this alone is by no means a guarantee of accurate findings. We discuss the important analytical challenges in quantifying 3-nitrotyrosine, possible workarounds, and we attempt to make sense of the disparate findings that have been reported so far.

Does Oxidative Stress Change During Orthotopic Liver Transplantation?

“…Admittedly, generation and establishment of new concepts are formidable challenges and Sisyphean tasks. Regarding the analytical approaches, mass spectrometry is the most promising technique; however, it is not free of pitfalls (5) and must be used correctly both for low-molecular-mass and high-molecular-mass biomolecules (28). In this work, we focused on OLT.…”

“…In patients suffering from end-stage liver disease, we measured higher concentrations of 15(S)-8-iso-prostaglandin F 2α (15(S)-8-iso-PGF 2α ) (2) and 3-nitrotyrosine and 3-nitrotyrosinoalbumin (3) than in healthy subjects. 15(S)-8-iso-PGF 2α and other F 2 -isoprostanes and 3-nitrotyrosine are generally considered reliable biomarkers of oxidative stress (lipid peroxidation) (1,4) and nitrosative stress (5), respectively. On the basis of the general assumption that elevated biomarker concentrations in biological samples are indicative of elevated oxidative and nitrosative stress, our findings on 15(S)-8-iso-PGF 2α , 3-nitrotyrosine and 3-nitrotyrosinoalbumin (2,3) would suggest that liver diseases are associated with elevated oxidative and jcbmr.com nitrosative stress.…”

“…On the basis of the general assumption that elevated biomarker concentrations in biological samples are indicative of elevated oxidative and nitrosative stress, our findings on 15(S)-8-iso-PGF 2α , 3-nitrotyrosine and 3-nitrotyrosinoalbumin (2,3) would suggest that liver diseases are associated with elevated oxidative and jcbmr.com nitrosative stress. However, oxidative and nitrosative stress represent a considerable challenge in this area of research (1,5), not least because both enzymatic and non-enzymatic sources including antioxidants such as reduced glutathione (GSH) commonly contribute to oxidative and nitrosative stress to a variable mostly of non-determinable extent. Thus, cyclooxygenase (6, 7) and myeloperoxidase are considerable contributors to 15(S)-8-iso-PGF 2α and 3-nitrotyrosine, respectively.…”

Tailored 96-well μElution solid-phase extraction combined with UFLC-MS/MS: a significantly improved approach for determination of free 3-nitrotyrosine in human urine

“…However, the accurate determination of 3-NT remains a daunting challenge due to severe methodological problems. High levels of 3-NT have been found in biological samples using methods lacking in sensitivity or/and specificity, so these results are suggested to be questionable or erroneous [6][7][8]. False overestimation may have mistakenly implicated 3-NT as a biomarker for oxidative damage and its biochemistry under diverse pathological conditions.…”

Nitrosative stress does not change during orthotopic liver transplantation

“…4 NT and NTALB are formed through the reaction of peroxynitrite with free tyrosine (Tyr) and Tyr moieties in albumin, respectively, and they serve as biomarkers of oxidative stress. 5 In comparison with age-and sex-matched healthy subjects, the patients who suffered from end-stage liver disease and underwent OLT had higher baseline plasma concentrations of NT (3.99 6 0.20 versus 1.14 6 0.21 nmol/L) and NTALB (27.7 6 1.2 versus 13.1 6 1.8 lmol of NT/mol of Tyr). Figure 1 shows that neither NT nor NTALB plasma concentrations changed statistically significantly during OLT.…”