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.
Public Health Relevance Statement: Thiol signaling by reactive nitrogen and oxygen species regulates aspects of tumor growth, migration, invasion, survival, angiogenesis, and metastasis. Redox sensitive cysteines in numerous cancer oncoproteins and tumor suppressor proteins have been identified by mutation analysis, though a technological void has prevented quantitation and characterization of their oxidation in a cellular context. Our proposal is to develop the OxMRM technology which combines protein enrichment with sensitive chemical analysis in order to quantify oxidation in potentially any cysteine for even low abundance proteins for the purposes of both basic research and in vivo analysis including diagnostic assays of clinical samples.
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