ID OF OXIDANT SENSITIVE CYSTEINE CONTAINING PROTEINS BY MASS SPECTROMETRY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们开发了一种使用半胱氨酸特异的、可酸裂解的同位素编码亲和标记(ICAT)试剂(应用生物系统，加利福尼亚州福斯特市)来鉴定和定量氧化剂敏感的蛋白质硫醇的方法。我们现在正在使用这种方法来探索单个半胱氨酸残基的氧化还原敏感性与生理上显著的氧化翻译后修饰之间的关系，以及与疾病相关的氧化应激导致的不可逆硫醇氧化之间的关系。我们已经定量评估了伴随其活性变化的重组蛋白H-RAS的氧化翻译后修饰。 H-RAS的氨基酸序列含有6个半胱氨酸残基。根据结构、化学和突变研究，在这六种病毒中，有四种(118、181、184和186)是表面暴露的。尽管已知Cys-181、Cys-184和Cys-186在完整细胞中可以通过预烯基化修饰，但在正常和病理条件下，所有反应性半胱氨酸都可能被氧化，这种氧化可能会改变蛋白质的细胞功能。我们已经证明RAS是由血管紧张素II刺激的平滑肌细胞中的NADPH氧化酶产生的氧化剂激活的。这一结果使得定量研究与其氧化剂介导的激活相关的蛋白质中的硫醇修饰是非常必要的。暴露于过氧亚硝酸根和谷胱甘肽后，RAS活性显著增加2~3倍，但不单独作用于过氧亚硝酸根。因此，我们应用我们的ICAT方法来识别和量化在存在和不存在谷胱甘肽的情况下使用ONOO治疗时发生的半胱氨酸修饰。ICAT标记的H-RAS多肽的MALDI-TOF MS显示有15-20个ICAT标记的多肽，其中轻的和重的ICAT标记的多肽有适当的9-Da差异。根据ICAT标记的重肽信号强度的变化，用LC-MS定量半胱氨酸的氧化程度。在ONOO处理的样品中，根据重标记多肽的强度变化测量，Cys-118被氧化了47%，而非反应性半胱氨酸80没有被氧化，这表明重ICAT标记多肽的强度没有变化。我们预计，对单个半胱氨酸残基修饰程度的定量评估可以与H-RAS在受到活性氧/氮物种作用时的激活或失活相关。因此，我们已经成功地应用我们的ICAT方法来定量评估伴随其活性变化的蛋白质H-RAS的氧化PTM。我们正在将这一方法扩展到其他已知(或被认为)在与心血管疾病相关的氧化应激中发挥重要作用的蛋白质。包括Sirt1。开发了一种高效表达Sirt1的方法，并产生了蛋白质和选择性修饰形式，从而可以确定结构/活性关系。使用双标记策略绘制修改后的位置图。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed an approach for identifying and quantifying oxidant-sensitive protein thiols using a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA). We are now using this approach to explore the relationship between redox sensitivity of individual cysteine residues and physiologically significant oxidative post-translational modifications, as well as irreversible thiol oxidation by oxidant stress associated with disease. We have quantitatively evaluated oxidative post-translational modifications of the recombinant protein H-Ras that accompany changes in its activity. The amino acid sequence of H-Ras contains six cysteine residues. Of the six, four (118, 181, 184 and 186) are surface-exposed, as determined by structural, chemical and mutational studies. Although Cys-181, Cys-184 and Cys-186 are known to be modified by prenylation in intact cells, all of the reactive cysteines are potentially oxidized during normal and pathological conditions and this oxidation could alter the cellular function of the protein. We have demonstrated that Ras was S-glutathiolated and activated by oxidants generated from NADPH oxidase in smooth muscle cells stimulated with angiotensin II. This result now makes it imperative to quantify the thiol modifications in the protein associated with its oxidant-mediated activation. The activity of Ras was significantly increased 2- to 3-fold following exposure to peroxynitrite and glutathione, but not to peroxynitrite alone. We therefore applied our ICAT approach to identify and quantify cysteine modifications that occur upon treatment with ONOO- in the presence and absence of glutathione. MALDI-TOF MS of the ICAT-labeled peptides of H-Ras showed 15-20 ICAT-labeled peptides with the appropriate 9-Da difference between the light and heavy ICAT-labeled peptides. LC-MS was used to quantify the degree of cysteine oxidation on the basis of the change in signal intensity for the heavy ICAT-labeled peptide. In the ONOO--treated samples, the Cys-118 is oxidized 47%, as measured from the change in the intensity for the heavy-labeled peptide, whereas the non-reactive Cys 80 is not oxidized as indicated by no change in the intensity for the heavy ICAT-labeled peptide. We anticipate that quantitative evaluation of the extent of modification of individual cysteine residues can be correlated to the activation or inactivation of H-Ras when subjected to reactive oxygen/nitrogen species. We have thus successfully applied our ICAT approach to quantitatively evaluate the oxidative PTMS of the protein H-Ras that accompany changes in its activity. We are extending this approach to include other proteins that are known (or thought) to play important roles in oxidative stress related to cardiovascular disease. including Sirt1. A method for efficient expression of Sirt1 was developed and the protein and selectively modified forms were generated so that structure/activity relationships could be determined. Modified sites were mapped using a double-labelling strategy.