Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.

Ksenija Vujacic-Mirski,Sebastian Steven,Bato Korac,Milos Mojovic,Swenja Kröller-Schön,Thomas Münzel,Sanela Kalinovic,Matthias Oelze,Kai Bruns,Andreas Daiber

doi:10.3390/antiox9050388

Abstract

Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•–) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected.

Highlights

Oxidative stress is reported to be a hallmark of almost all neurodegenerative and cardiovascular diseases [1,2,3]
Cellular oxidative stress conditions are defined by the increased formation of reactive oxygen and nitrogen species and/or impaired cellular antioxidant defense system, depletion of low molecular weight antioxidants, and a shift in the cellular redox balance [6,7], which is associated with oxidative damage of biomolecules such as proteins [8,9]
Digested samples were used for the detection by high performance liquid chromatography (HPLC)/ECD while parts of undigested samples were used for the detection by dot blot analysis or enzyme-linked immunosorbent assay (ELISA)

Summary

Introduction

Oxidative stress is reported to be a hallmark of almost all neurodegenerative and cardiovascular diseases [1,2,3]. A prominent example is the nitration, e.g., by peroxynitrite (PN) of Tyr in mitochondrial superoxide dismutase (MnSOD) [10,11,12], which is associated with its inhibition and the pathogenesis of various diseases [13,14,15]. There is increasing evidence that redox modifications of proteins can affect enzyme activities and represent alterations of the cellular signaling network (reviewed in [16,17,18,19]). Protein tyrosine nitration represents a prominent posttranslational redox modification and is associated with a broad range of different diseases [3,20,21]. A similar metal-catalyzed mechanism was postulated for MnSOD that facilitates PN-mediated nitration and dimerization of tyrosine residues, leading to inactivation of the enzyme [10,11,12].

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