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

• 批准号: 8365499
• 负责人: RICHARD A COHEN
• 金额: $ 0.77万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365499
• 关键词: Acids; Am 80; Amino Acid Sequence; Angiotensin II; Biology; Cardiovascular Diseases; Cell physiology; Cells; Chemicals; Cities; Cysteine; Disease; Exposure to; Fostering; Funding; Glutathione; Grant; HRAS gene; Individual; Isotopically-Coded Affinity Tagging; Label; Light; Maps; Mass Spectrum Analysis; Measures; Mediating; Medicine; Methods; Modification; NADPH Oxidase; National Center for Research Resources; Nitrogen; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Peptides; Peroxonitrite; Play; Post-Translational Protein Processing; Principal Investigator; Proteins; Quantitative Evaluations; Reagent; Recombinant Proteins; Research; Research Infrastructure; Resources; Role; Sampling; Signal Transduction; Site; Smooth Muscle Myocytes; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship; Sulfhydryl Compounds; Surface; United States National Institutes of Health; base; c-Ha-ras p21; cost; oxidant stress; oxidation; prenylation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 are being mapped and the modifications are being determined.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们已经开发了一种使用半胱氨酸特异性的，可辨认的同位素编码亲和含量标签（ICAT）试剂（Applied Biosystems，CA）的方法来识别和量化氧化剂敏感蛋白硫醇。现在，我们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重要的氧化后翻译后修饰，以及与疾病相关的氧化应激的不可逆硫醇氧化。我们已经定量评估了伴随其活性变化的重组蛋白H-RAS的氧化后翻译后修饰。 H-RAS的氨基酸序列包含六个半胱氨酸残基。在六个（118、181、184和186）中，在结构，化学和突变研究中确定了表面暴露。尽管已知CYS-181，CYS-184和CYS-186是通过完整细胞中的婚前化来改变的，但在正常和病理条件下，所有反应性半胱氨酸都可能氧化，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是由血管紧张素II刺激的平滑肌细胞中NADPH氧化酶产生的氧化剂S-glutathiol和激活的。现在，该结果必须量化与其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的活性显着增加了2至3倍，但单独使用过氧亚硝酸盐。因此，我们应用了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-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标记的肽，在光和重型ICAT标记的肽之间具有适当的9-DA差异。 LC-MS用于根据重ICAT标记的肽的信号强度的变化来量化半胱氨酸氧化的程度。在经过处理的样品中，Cys-118被氧化了47％，这是根据重标记肽的强度的变化所测量的，而非反应性CYS 80并未通过未通过强度的变化来氧化重的ICAT标记肽的强度。我们预计，当受到活性氧/氮种时，对单个半胱氨酸残基修饰程度的定量评估可以与H-RAS的激活或灭活相关。因此，我们已经成功地应用了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。我们将这种方法扩展到包括其他已知（或认为）的蛋白质，以在与心血管疾病有关的氧化应激中起重要作用。包括SIRT1。开发了一种有效表达的方法，并生成了蛋白质和选择性修改的形式，以便可以确定结构/活动关系。正在映射修改的站点，并确定修改。