Site-specific quantitation of protein nitration using liquid chromatography/tandem mass spectrometry
Site-specific quantitation of protein nitration using liquid chromatography/tandem mass spectrometry
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DOI：
10.1021/ac034033j
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发表时间：
2003-05-15
影响因子：
7.4
通讯作者：
Kinter, M
中科院分区：
文献类型：
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作者：
Willard, BB;Ruse, CI;Kinter, M
新的天然参比肽（NRP）法适用于测定特定酪氨酸残基上的蛋白质硝化程度。在这些实验中，在亚硝酸盐存在下，人血清白蛋白在髓过氧化物酶介导的反应中被修饰，其中主要在Y-162位点检测到硝化作用。根据肽（162）Y(n)LYEIAR（168）丰度的增加和凝胶胰酶消化中（162）YLYEIAR（168）肽的相应减少来测量该位点的硝化作用的时间依赖性增加。肽（66）LVNEVTEFAK（75）也在胰蛋白酶消化中形成，被用作天然参考肽。定量通过LC/串联质谱分析确定两种分析肽相对于天然参比肽的色谱峰面积，并选择反应监测。NRP结果与Western blot测定的总蛋白-硝基酪氨酸含量随时间增加的相关性进行了验证。对该方法的精密度和检出限进行了评估，发现其柱上含量分别约为10%（相对标准偏差）和5 fmol。这些结果证明了NRP方法在翻译后修饰定量分析方面的实用性，包括广泛的适用性、易于实验设计、灵敏度和精度。
ne 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, Y-162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide (162)Y(n)LYEIAR(168) and the corresponding decrease in the (162)YLYEIAR(168) peptide in in-gel trypsin digests. The peptide (66)LVNEVTEFAK(75), 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.