Stable isotope metabolic labeling-based quantitative thiol redox proteomic analysis of hydrogen peroxide-treated Arabidopsis plant

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Abstract

Homeostasis of protein thiol redox plays an important role in numerous cellular processes and stress responses. The thiol moiety of cysteine residue is easily altered to various redox isoforms that play critical roles in regulation of both structure and function of proteins. In the present study, a quantitative thiol redox proteomic method, termed OxNSIL, has been developed, which integrates the chemical labeling by biotin-tagged alkylating reagents with the heavy nitrogen stable isotope-coded salt metabolic labeling and acquires both advantages of biotin-avidin enrichment and MS-based quantitation of cysteine residue-containing peptides. Both the reduced and the reversibly oxidized cysteine thiol moieties are finally identified in a site-specific manner and measured in a single experiment. Hydrogen peroxide, H2O2, was applied on the whole Arabidopsis plant to trigger thiol redox state alteration in order to obtain a proof-of-concept result for application of OxNSIL approach in plants. A total of 438 non-redundant biotin-tagged thiolalkylated peptides representing 391 different cysteine-containing proteins were eventually obtained, among which 17 redox-sensitive biotin tag-labeled peptides were significantly altered by H2O2. Some well-known ROS-related proteins, such as Ferredoxins 1/2, RuBisCO large subunit, RuBisCO activase, and Fructose-bisphosphate aldolase 6/8 were also detected as the oxidation- sensitive thiol redox proteins. These results substantiate the usefulness of OxNSIL approach in study of in planta ROS-induced cellular thiol redox state alterations. © 2014 Hu Q, et al.

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Hu, Q., Guo, G., Yang, Z., Li, Y., Xia, Y., & Li, N. (2014). Stable isotope metabolic labeling-based quantitative thiol redox proteomic analysis of hydrogen peroxide-treated Arabidopsis plant. Journal of Proteomics and Bioinformatics, 7(5), 121–133. https://doi.org/10.4172/jpb.1000312

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