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。作为原理的证明，我们定量地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(1-10微克)用过氧亚硝酸根(ONOO-，100微米)处理5分钟。在37℃存在或不存在还原型谷胱甘肽的情况下。RAS活性测定采用Raf-1结合法和GTP/GDP交换法。ONOO处理的样品在37℃孵育30min，用重ICAT试剂标记。将重同位素标记的蛋白质与未经处理的H-RAS等量混合，这些H-RAS之前曾用轻质ICAT试剂标记。轻标记和重标记蛋白用胰酶消化，脱盐，并使用亲和素盒(美国应用生物系统公司)进行亲和纯化。样品经浓缩后用MALDI-TOF MS和CapLC-MS/MS进行分析。 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。我们最近证实，血管紧张素II刺激的血管平滑肌细胞中，RAS是由NADPH氧化酶产生的氧化剂激活的。这一结果使得我们有必要对与其氧化剂介导的激活相关的蛋白质中的硫醇修饰进行定量。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。已经开发了一种高效表达Sirt1的方法，并且正在产生蛋白质和选择性修饰形式，以便能够确定结构/活性关系。

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. As a proof of principle, we have quantitatively evaluated oxidative post-translational modifications of the recombinant protein H-Ras that accompany changes in its activity. H-Ras (1-10 ug) was treated with peroxynitrite (ONOO-, 100 uM) for 5 min. at 37 deg C in the presence or absence of reduced glutathione. Ras activity was assayed by association with Raf-1 and by GTP/GDP exchange. The ONOO--treated samples were labeled with heavy ICAT reagent by incubating at 37 deg C for 30 min. The heavy isotope-labeled protein was mixed in equal amounts with untreated H-Ras that had previously been labeled with light ICAT reagent. The light and heavy labeled proteins were digested with trypsin, desalted, and affinity-purified using an avidin cartridge (Applied Biosystems, USA). The samples were concentrated and analyzed by MALDI-TOF MS and capLC-MS/MS. 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 recently 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 has been developed and the protein and selectively modified forms are being generated so that structure/activity relationships can be determined.