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Quantification of 3-nitrotyrosine levels using a benchtop ion trap mass spectrometry method
Stephen J Nicholls1, Zhongzhou Shen, Xiaoming Fu
1Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Department of Cell Biology, Cleveland, OH 44195, USA.
Methods in Enzymology
|November 18, 2005
Summary
Researchers developed a new method to quantify 3-Nitrotyrosine (NO2Tyr), a marker of oxidative damage. This technique aids in understanding reactive nitrogen species
Area of Science:
- Biochemistry
- Analytical Chemistry
- Pathophysiology
Background:
- Oxidative damage from reactive nitrogen species (RNS) contributes to inflammatory diseases like atherosclerosis.
- 3-Nitrotyrosine (NO2Tyr) is a biomarker for RNS-mediated protein modification and a predictor of cardiovascular risk.
- Accurate quantification of NO2Tyr in biological samples is crucial for clinical and research applications.
Purpose of the Study:
- To develop a rapid, sensitive, and specific analytical method for quantifying NO2Tyr in biological matrices.
- To establish a reliable method for assessing nitrative stress and cardiovascular risk.
Main Methods:
- Utilized high-performance liquid chromatography (HPLC) coupled with benchtop ion-trap mass spectrometry.
- Employed stable isotopically labeled precursors as internal standards for accurate quantification.
- The tandem mass spectrometry approach corrected for sample loss and artifactual NO2Tyr generation.
Main Results:
- Achieved a detection limit of 100 fmol on column for NO2Tyr.
- Demonstrated a rapid and reproducible quantification method for biological and clinical specimens.
- The method effectively adjusts for sample preparation variables.
Conclusions:
- The developed HPLC-MS/MS method provides a robust tool for NO2Tyr quantification.
- This technique can be valuable for studying the role of RNS in cardiovascular disease and inflammatory disorders.
- NO2Tyr measurement offers a potential prognostic marker for clinical relevance.