ID OF OXIDANT SENSITIVE CYSTEINE CONTAINING PROTEINS BY MASS SPECTROMETRY

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 我们已经开发了一种使用半胱氨酸特异性、酸可裂解的同位素编码的亲和标签（ICAT）试剂（Applied Biosystems，Foster City，CA）鉴定和定量氧化剂敏感性蛋白硫醇的方法。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和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引起的不可逆巯基氧化。作为一个原则的证明，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。在存在或不存在还原型谷胱甘肽的情况下，将H-Ras（1-10 μ g）用过氧亚硝酸盐（ONOO-，100 μ M）在37 ℃下处理5分钟。Ras活性通过与Raf-1的结合和GTP/GDP交换来测定。通过在37 ℃下孵育30分钟，用重ICAT试剂标记ONOO-处理的样品。将重同位素标记的蛋白质与先前用轻ICAT试剂标记的未处理的H-Ras等量混合。用胰蛋白酶消化轻和重标记的蛋白质，脱盐，并使用抗生物素蛋白柱（Applied Biosystems，USA）亲和纯化。将样品浓缩并通过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6个基因中，有4个（118、181、184和186）是表面暴露的。尽管已知Cys-181、Cys-184和Cys-186在完整细胞中通过异戊二烯化修饰，但所有反应性半胱氨酸在正常和病理条件下都可能被氧化，并且这种氧化可能改变蛋白质的细胞功能。我们最近证明，Ras是S-谷胱甘肽和激活的氧化剂产生的NADPH氧化酶在血管紧张素II刺激平滑肌细胞。这一结果现在使得量化与其氧化剂介导的活化相关的蛋白质中的巯基修饰势在必行。Ras的活性显着增加2- 3倍暴露后过氧亚硝酸盐和谷胱甘肽，但不单独过氧亚硝酸盐。因此，我们应用我们的ICAT方法来鉴定和定量在存在和不存在谷胱甘肽的情况下用ONOO处理后发生的半胱氨酸修饰。H-Ras的ICAT标记肽的MALDI-TOF MS显示15-20个ICAT标记肽，轻和重ICAT标记肽之间具有适当的9-Da差异。基于重ICAT标记肽的信号强度变化，使用LC-MS定量半胱氨酸氧化程度。在ONOO-处理的样品中，Cys-118被氧化47%，如从重标记肽的强度变化所测量的，而非反应性Cys 80没有被氧化，如重ICAT标记肽的强度没有变化所指示的。我们预计，个别半胱氨酸残基的修饰程度的定量评价可以与活化或失活的H-Ras时，受到活性氧/氮物种。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PTMS。我们正在扩展这种方法，以包括其他已知（或认为）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Sirt 1。已经开发了一种有效表达Sirt 1的方法，并且正在产生蛋白质和选择性修饰形式，以便可以确定结构/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.