| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 101 === Distinctive states of redox-dependent cysteine (Cys) modifications, such as sulfenation (SOH), S-nitrosylation (SNO) and S-glutathionylation (SSG), are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. Simultaneous monitoring of the dynamics of various reversible cysteine modifications is required to obtain a systems view of how cells response to changing redox status. However, a precise delineation of multiplexed Cys-modifications in a cellular context remains technically challenging. In this thesis, we reported the development, optimization, and applications of mass spectrometry (MS)-based quantitative approaches for global and targeted redox proteomic studies that mostly based on sequential cycles of selective reduction and irreversible alkylation with isotope-coded or isobaric mass tags endowed with Cys-reactivities. By applying the sequential alkylation switch workflow, all Cys thiols in the samples were differentially labeled with designated tags depending on their initial redox status and were then quantitatively measured by following MS analysis with or without immunoenrichment. We first demonstrated the capabilities of this approach by proving the accurate and site-specific measurement of the fold change and the stoichiometry of multiplexed reversible Cys modifications on purified PTP1B treated with S-nitrosoglutathione (GSNO). Next, we applied the workflow to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. In one single analysis, we have identified over 260 Cys sites showing quantitative differences in multiplexed redox-modifications from the total lysates of H9c2 cardiomyocytes experiencing hypoxia in the absence and presence of GSNO, indicative of a distinct pattern of individual susceptibility to S-nitrosylation or S-glutathionylation. Among those most significantly affected are proteins functionally implicated in hypoxic damage from which we showed that GSNO would protect. We thus demonstrate for the first time how quantitative analysis of various Cys-redox modifications occurring in biological samples can be performed precisely and simultaneously at proteomic levels. Finally, selected reaction monitoring (SRM) assays coupled with complete immunoprecipitation were developed to quantitatively follow the dynamics of Cys modifications on targeted proteins as effected through external NO donors and/or physiologically relevant stimuli. We showed that, by this targeted approach, significant increase of SNO was occurred site-specifically on catalytic Cys of endogenous SHP-2 in NO-protected cardiomyocyte under hypoxic insult, which was not detectable in global redox-proteomic analysis. In conclusion, we have not only developed a new approach to map global Cys-redoxomic regulation in vivo, but also provided new evidences implicating Cys-redox modifications of key molecules in NO-mediated ischemic cardioprotection.
|
|---|
