Nicholls Stephen J, Shen Zhongzhou, Fu Xiaoming, Levison Bruce S, Hazen Stanley L
Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Department of Cell Biology, Cleveland, OH 44195, USA.
Methods Enzymol. 2005;396:245-66. doi: 10.1016/S0076-6879(05)96022-9.
Oxidative damage by reactive nitrogen species is linked to the pathogenesis of numerous inflammatory disorders, including atherosclerosis. 3-Nitrotyrosine (NO2Tyr), a posttranslational modification of proteins generated by reactive nitrogen species, serves as a "molecular fingerprint" for protein modification by nitric oxide (NO)-derived oxidants. Studies demonstrate that systemic levels of protein-bound NO2Tyr serve as an independent predictor of cardiovascular risks and are modulated by statin therapy. Measurement of NO2Tyr in biological matrices may thus serve both as a quantitative index of nitrative stress in vivo and an important new prognostic marker of clinical relevance. Analytical methods for the accurate detection and quantification of trace levels of NO2Tyr in biological tissues and fluids are, thus, of considerable interest. Here, we describe a rapid, sensitive, and specific method for the quantification of NO2Tyr in biological matrices using readily available benchtop ion-trap mass spectrometry instrumentation (e.g., LCQDeca) combined with high-performance liquid chromatography (HPLC) interface. Through judicious use of stable isotopically labeled precursors as synthetic internal standards, the tandem mass spectrometric method described simultaneously adjusts for potential intrapreparative sample losses and monitors potential artifactual generation of NO2Tyr during processing. The described method permits rapid and reproducible quantification of NO2Tyr in biological and clinical specimens at the 100 fmol on column detection limit and should prove useful for studies defining the impact of reactive nitrogen species in cardiovascular disease and other inflammatory disorders.
活性氮物质引起的氧化损伤与包括动脉粥样硬化在内的多种炎症性疾病的发病机制有关。3-硝基酪氨酸（NO2Tyr）是由活性氮物质产生的蛋白质翻译后修饰产物，可作为一氧化氮（NO）衍生氧化剂对蛋白质修饰的“分子指纹”。研究表明，与蛋白质结合的NO2Tyr的全身水平可作为心血管风险的独立预测指标，并受他汀类药物治疗的调节。因此，在生物基质中测量NO2Tyr既可以作为体内硝化应激的定量指标，也可以作为具有临床相关性的重要新预后标志物。因此，能够准确检测和定量生物组织和体液中痕量水平NO2Tyr的分析方法备受关注。在此，我们描述了一种使用易于获得的台式离子阱质谱仪器（例如LCQDeca）结合高效液相色谱（HPLC）接口来定量生物基质中NO2Tyr的快速、灵敏且特异的方法。通过明智地使用稳定同位素标记的前体作为合成内标，所描述的串联质谱方法可同时校正制备过程中潜在的样品损失，并监测处理过程中NO2Tyr的潜在人为生成。所描述的方法能够在柱上检测限为100 fmol时对生物和临床样本中的NO2Tyr进行快速且可重复的定量，并且对于确定活性氮物质在心血管疾病和其他炎症性疾病中的影响的研究应该是有用的。
