Willard Belinda B, Ruse Cristian I, Keightley J Andrew, Bond Meredith, Kinter Michael
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Anal Chem. 2003 May 15;75(10):2370-6. doi: 10.1021/ac034033j.
The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, Y162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide 162YnLYEIAR168 and the corresponding decrease in the 162YLYEIAR168 peptide in in-gel trypsin digests. The peptide 66LVNEVTEFAK75, also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.
天然参考肽（NRP）方法已被用于测量特定酪氨酸残基处蛋白质硝化的程度。在这些实验中，人血清白蛋白在亚硝酸盐存在下通过髓过氧化物酶介导的反应进行修饰，硝化主要发生在一个位点Y162。基于凝胶内胰蛋白酶消化物中肽162YnLYEIAR168丰度的增加以及162YLYEIAR168肽相应的减少，测量该位点硝化随时间的增加。胰蛋白酶消化物中形成的肽66LVNEVTEFAK75也用作天然参考肽。通过使用选择反应监测的液相色谱/串联质谱分析，通过确定两种分析物肽相对于天然参考肽的色谱峰面积来实现定量。通过与蛋白质免疫印迹分析确定的总蛋白质硝基酪氨酸含量随时间的增加进行相关性分析，验证了NRP结果。还评估了该测定法的精密度和检测限，发现分别约为10%（相对标准偏差）和柱上5 fmol。这些结果证明了NRP方法在翻译后修饰定量分析中的实用性，体现在广泛的适用性、易于实验设计、灵敏度和精密度方面。
