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

描述（由申请人提供）：活性氮和氧物质（RN/OS）的巯基信号传导调节肿瘤生长、迁移、侵袭、存活、血管生成和转移的各个方面;然而，癌症中巯基信号传导的关键修饰和机制相对模糊。尽管长期暴露于RNOS传统上被认为是有害的，但巯基信号传导对正常细胞功能至关重要，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还没有得到很好的理解，因为控制它们形成的化学物质通常是短暂和复杂的。半胱氨酸氧化分析的困难导致了技术空白，其中许多癌基因，如p53，NF？B和HIF 1a已知受关键巯基调节，但对其氧化状态知之甚少。此外，氧化调节的机制，以及它在癌症发生和发展过程中如何变化还不清楚。使用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异巯基烷基化、蛋白质纯化和多反应监测（MRM）分析来灵敏地定量潜在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灵敏和最定量的质谱（MS）技术，并且将允许对来自细胞和体内来源的甚至低水平的内源性蛋白质（例如p53）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的硫醇氧化信号传导与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。

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.