OxMRM: A Technique to Quantify Oxidation of Endogenous Redox-Sensitive Cysteines

OxMRM：一种量化内源性氧化还原敏感半胱氨酸氧化的技术

• 批准号: 7628935
• 负责人: Jason M. Held
• 金额: $ 25.61万
• 依托单位: BUCK INSTITUTE FOR RESEARCH ON AGING
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7628935
• 关键词: Acute; Address; Alkylation; Apoptosis; Apoptotic; Basic Science; Biological; Biological Assay; Cancer Biology; Cancerous; Cell physiology; Cells; Chemicals; Chemistry; Chronic; Clinical; Complex; Cysteine; Development; Diagnostic; Exposure to; Gene Mutation; Genes; Genetic Transcription; Induction of Apoptosis; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Modeling; Modification; Mutation Analysis; Neoplasm Metastasis; Nitrogen; Oncogene Proteins; Oncogenes; Oxidation-Reduction; Oxidative Stress; Oxygen; Phosphotransferases; Play; Post-Translational Protein Processing; Predisposition; Premalignant; Production; Proteins; Reactive Nitrogen Species; Reactive Oxygen Species; Reagent; Regulation; Role; Sampling; Signal Transduction; Source; Stable Isotope Labeling; Stress; Sulfhydryl Compounds; System; TP53 gene; Techniques; Technology; Transactivation; Tumor Suppressor Proteins; Work; angiogenesis; anti-cancer therapeutic; cancer initiation; carcinogenesis; in vivo; insight; migration; multiple reaction monitoring; neoplastic cell; oxidation; prevent; promoter; protein purification; public health relevance; repaired; response; tumor growth; tumor progression

DESCRIPTION (provided by applicant): Thiol signaling by reactive nitrogen and oxygen species (RN/OS) regulates aspects of tumor growth, migration, invasion, survival, angiogenesis, and metastasis; however the key modifications and mechanisms of thiol signaling in cancer are relatively obscure. Although chronic exposure to RNOS has traditionally been thought to be deleterious, thiol signaling is essential for normal cellular function, suggesting that RNOS play a complex role in cancer biology. Insights into cysteine oxidation signaling in cancer will further our understanding of cancer progression, as well as aid development of new anti-cancer therapeutics. A variety of RNOS molecules oxidize thiols leading to a diverse array of modifications. The biological consequences of these modifications, however, are not well understood, since the chemistry that governs their formation is often transient and complex. The difficulty of cysteine oxidation analysis has led to a technological void in which the function of many oncogenes such as p53, NF?B, and HIF1a are known to be regulated by key thiols, though little is known about their oxidation status. In addition, the mechanism by which oxidation is regulated, and how it changes during cancer initiation and progression are not well understood. Using p53 as a model redox sensitive protein, this study will address this challenge by proposing a technology, (OxMRM), capable of sensitively quantifying cysteine oxidation status of potentially any protein by integrating differential thiol alkylation, protein purification, and analysis by multiple reaction monitoring (MRM). The advantage of MRM is that it is the most sensitive and quantitative mass spectrometry (MS) technique available, and will allow thiol oxidation analysis of even low level endogenous proteins such as p53 from both cellular and in vivo sources. OxMRM can address the interface between essential thiol oxidation signaling and the chronic effects of increased RNOS production by examining the reversible redox status of proteins as well as potentially irreversible oxidation. This study aims to uncover insights into the functional consequences of oxidation and whether, in the case of p53, disruption leads to increased susceptibility to DNA mutations.

描述（由申请人提供）：反应性氮和氧（RN/OS）的硫醇信号传导调节肿瘤生长，迁移，侵袭，生存，血管生成和转移的各个方面；但是，癌症中硫醇信号的关键修饰和机制相对晦涩。尽管传统上认为长期暴露于RNO是有害的，但硫醇信号对于正常的细胞功能至关重要，这表明RNO在癌症生物学中起着复杂的作用。对癌症中半胱氨酸氧化信号传导的见解将进一步了解我们对癌症进展的理解，并有助于开发新的抗癌疗法。多种RNO分子氧化硫醇会导致各种修饰。然而，这些修饰的生物学后果尚不清楚，因为控制它们的形成的化学反应通常是短暂而复杂的。半胱氨酸氧化分析的难度导致了一种技术空隙，其中许多肿瘤基因（例如p53，nf？b和hif1a）的功能受到关键硫醇的调节，尽管对其氧化状态知之甚少。另外，对氧化的调节机制以及在癌症开始和进展过程中的变化方式尚不清楚。该研究将使用p53作为模型氧化还原敏感蛋白，通过提出技术（OXMRM）来解决这一挑战，该技术能够通过整合差异硫醇烷基化，蛋白质纯化，蛋白质纯化，并通过多个反应监测（MRM）来敏感地量化任何蛋白质的半胱氨酸氧化状态。 MRM的优点是它是可用的最敏感和定量质谱（MS）技术，并且可以允许对均匀水平的内源性蛋白（例如细胞和体内源）进行硫醇氧化分析。 OXMRM可以通过检查蛋白质的可逆氧化还原状态以及潜在的不可逆氧化来解决必需硫醇氧化信号传导与增加RNOS产生的慢性影响之间的界面。这项研究的目的是发现氧化功能后果以及在p53的情况下是否导致对DNA突变的敏感性增加。