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RESEARCH PRODUCT
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.
Bato KoracMatthias OelzeAndreas DaiberKsenija Vujacic-mirskiThomas MünzelKai BrunsSanela KalinovicMiloš MojovićSebastian StevenSwenja Kröller-schönsubject
0301 basic medicinePhysiologyClinical BiochemistryDot blotmitochondrial superoxidePronase030204 cardiovascular system & hematologymedicine.disease_causeBiochemistryHigh-performance liquid chromatographyArticleperoxynitritePeroxynitrite03 medical and health sciences0302 clinical medicineProtein-bound 3-nitrotyrosinemedicineoxidative stressBovine serum albuminMolecular Biologychemistry.chemical_classificationDetection limitReactive oxygen speciesChromatographyHPLC with electrochemical detectionbiologylcsh:RM1-950Cell Biology3. Good health030104 developmental biologylcsh:Therapeutics. PharmacologychemistryOxidative stressbiology.proteinprotein-bound 3-nitrotyrosineOxidative stressEx vivoMitochondrial superoxidedescription
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&bull
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2020-05-01 |