ID OF OXIDANT SENSITIVE CYSTEINE CONTAINING PROTEINS BY MASS SPECTROMETRY

通过质谱法鉴定含氧化剂敏感半胱氨酸的蛋白质

基本信息

批准号：
8170862
负责人：
RICHARD A COHEN
金额：
$ 0.93万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8170862
关键词：
Acids Am 80 Amino Acid Sequence Angiotensin II Cardiovascular Diseases Cell physiology Cells Chemicals Cities Computer Retrieval of Information on Scientific Projects Database Cysteine Disease Exposure to Fostering Funding Glutathione Grant HRAS gene Individual Institution Isotopically-Coded Affinity Tagging Label Light Maps Mass Spectrum Analysis Measures Mediating Methods Modification NADPH Oxidase Nitrogen Oxidants Oxidation-Reduction Oxidative Stress Oxygen Peptides Peroxonitrite Play Post-Translational Protein Processing Proteins Quantitative Evaluations Reagent Recombinant Proteins Research Research Personnel Resources Role Sampling Signal Transduction Site Smooth Muscle Myocytes Source Structure-Activity Relationship Sulfhydryl Compounds Surface United States National Institutes of Health base c-Ha-ras p21 oxidant stress oxidation prenylation

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。我们开发了一种使用半胱氨酸特异性、酸可裂解同位素编码亲和标签 (ICAT) 试剂（Applied Biosystems，福斯特城，加利福尼亚州）来识别和定量氧化剂敏感蛋白硫醇的方法。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与生理上显着的氧化翻译后修饰以及与疾病相关的氧化应激引起的不可逆硫醇氧化之间的关系。我们定量评估了重组蛋白 H-Ras 伴随其活性变化的氧化翻译后修饰。 H-Ras的氨基酸序列包含六个半胱氨酸残基。根据结构、化学和突变研究确定，在这 6 个中，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进行修饰，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，并且这种氧化可能会改变蛋白质的细胞功能。我们已经证明 Ras 被 S-谷胱甘肽化，并被血管紧张素 II 刺激的平滑肌细胞中 NADPH 氧化酶产生的氧化剂激活。现在，这一结果使得必须量化与氧化剂介导的激活相关的蛋白质中的硫醇修饰。暴露于过氧亚硝酸盐和谷胱甘肽后，Ras 活性显着增加 2 至 3 倍，但单独接触过氧亚硝酸盐则不然。因此，我们应用 ICAT 方法来识别和量化在存在和不存在谷胱甘肽的情况下用 ONOO- 处理时发生的半胱氨酸修饰。 H-Ras 的 ICAT 标记肽的 MALDI-TOF MS 显示有 15-20 个 ICAT 标记肽，轻链和重链 ICAT 标记肽之间有适当的 9 Da 差异。 LC-MS 用于根据重 ICAT 标记肽的信号强度变化来量化半胱氨酸氧化程度。在 ONOO 处理的样品中，根据重标记肽的强度变化测量，Cys-118 被氧化了 47%，而非反应性 Cys 80 没有被氧化，强度没有变化表明对于重 ICAT 标记的肽。我们预计，当受到活性氧/氮物质影响时，对单个半胱氨酸残基修饰程度的定量评估可以与 H-Ras 的激活或失活相关。因此，我们成功地应用我们的 ICAT 方法来定量评估蛋白质 H-Ras 伴随其活性变化的氧化 PTMS。我们正在扩展这种方法，以包括已知（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其他蛋白质。包括 Sirt1。开发了一种有效表达 Sirt1 的方法，并生成了蛋白质和选择性修饰形式，从而可以确定结构/活性关系。使用双标记策略对修饰位点进行定位。