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: 7798156
• 负责人: Jason M. Held
• 金额: $ 29.88万
• 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/7798156
• 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）：硫醇信号通过活性氮和氧（RN/OS）调控肿瘤生长、迁移、侵袭、存活、血管生成和转移等方面；然而，硫醇信号在癌症中的关键修饰和机制相对不清楚。尽管传统上认为长期暴露于RNOS是有害的，但硫醇信号对于正常细胞功能是必不可少的，这表明RNOS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的深入了解将进一步加深我们对癌症进展的理解，并有助于开发新的抗癌疗法。各种RNOS分子氧化硫醇导致各种各样的修饰。然而，这些修饰的生物学后果尚不清楚，因为控制它们形成的化学反应往往是短暂而复杂的。半胱氨酸氧化分析的困难导致了技术空白，其中许多致癌基因如p53， NF？已知B和HIF1a受关键硫醇调节，但对其氧化状态知之甚少。此外，氧化被调节的机制，以及它在癌症发生和发展过程中是如何变化的还不是很清楚。利用p53作为模型氧化还原敏感蛋白，本研究将通过提出一种技术（OxMRM）来解决这一挑战，该技术能够通过整合差分巯基烷基化、蛋白质纯化和多重反应监测（MRM）分析，灵敏地定量任何蛋白质的半胱氨酸氧化状态。MRM的优势在于它是最敏感和定量的质谱（MS）技术，并且可以对细胞和体内来源的低水平内源性蛋白质（如p53）进行硫醇氧化分析。OxMRM可以通过检测蛋白质的可逆氧化还原状态以及潜在的不可逆氧化，解决必需硫醇氧化信号与RNOS产生增加的慢性效应之间的界面。这项研究旨在揭示氧化的功能后果，以及在p53的情况下，破坏是否会导致对DNA突变的易感性增加。