Quantitative proteomics of cellular protein-thiol redox homeostasis

2015 Fiscal Year Final Research Report

• Project/Area Number: 26750376
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Chemical biology
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: Araki Kazutaka 国立研究開発法人産業技術総合研究所, 創薬分子プロファイリング研究センター, 研究員 (60514255)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 質量分析測定 / タンパク質 / 酸化還元

Outline of Final Research Achievements

The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation, like ICAT method. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. Modest H2O2 treatment (50 μM for 5 min) did not cause global oxidations but, instead, had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins. Overall, we could succeed to yeild quantitative data, illustrating both H2O2- and reduction-mediated cellular responses.

Free Research Field

生物化学