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：
7601956
负责人：
RICHARD A COHEN
金额：
$ 1.51万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-03 至 2008-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7601956
关键词：
Acids Affinity Am 80 Amino Acid Sequence Angiotensin II Avidin Biological Assay Cardiovascular Diseases Cell physiology Cells Chemicals Cities Computer Retrieval of Information on Scientific Projects Database Condition Cysteine Disease Exposure to Fostering Funding Glutathione Grant HRAS gene Hares Incubated Individual Institution Isotope Labeling Isotopically-Coded Affinity Tagging Label Light Mass Spectrum Analysis Measures Mediating Modification NADPH Oxidase Nitrogen Oxidants Oxidation-Reduction Oxidative Stress Oxygen Peptides Peroxonitrite Play Post-Translational Protein Processing Proteins Quantitative Evaluations Reagent Recombinant Proteins Reduced Glutathione Research Research Personnel Resources Role Sampling Signal Transduction Smooth Muscle Myocytes Source Stress Sulfhydryl Compounds Surface Thinking Trypsin United States National Institutes of Health base c-Ha-ras p21 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. 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 oC 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 hare extending this approach to include other proteins that are known (or thought) to play important roles in oxidative stress related to cardiovascular disease.

该副本是利用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这不一定是调查员的机构。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。作为原则的证明，我们已经定量评估了伴随其活性变化的重组蛋白H-RAS的翻译后修饰。 H-RAS（1-10 UG）用过氧亚硝酸盐（ONOO-，100 UM）处理5分钟。在存在或不存在减少谷胱甘肽的情况下，在37°C处。通过与RAF-1和GTP/GDP交易所相关的RAS活动是通过与RAS活性分析的。通过在37 oC下孵育30分钟，用重型ICAT试剂标记了ONOO-ONOO-样品。将重量的同位素标记的蛋白与未处理的H-RAS相等地混合，该蛋白以前曾用轻质ICAT试剂标记。使用Avidin墨盒（Applied Biosystems，USA）用胰蛋白酶，脱盐和亲和纯化的光蛋白（美国，美国，美国）消化。通过MALDI-TOF MS和CAPLC-MS/MS浓缩样品并分析样品。 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。我们最近证明了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-谷氨酸促别洛别尔致激活的。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。