Development of a chemo-enzymatic method for probing protein glutathionylation

• Main Authors: Fu-Tan Hsieh, 謝馥檀
• Other Authors: 林俊宏
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/87945465617129819203

碩士 === 臺灣大學 === 生化科學研究所 === 98 === Protein S-glutathionylation is the formation of mixed disulfide bonds between the thiols of glutathione (GSH) and protein cysteine residues, representing a post-translational modification of proteins. This special modification protects protein thiols from irreversible oxidation and regulates protein functions. To identify S-glutathionylated proteins is the prerequisite to understand the physiolocical function, but the current progress is mainly restricted by currently available methods. Glutathionylspermidine synthetase (GspS) catalyzes the amide bond formation between GSH and spermidine to synthesize glutathionylspermidine (Gsp). Using GspS and synthesized spermidine-biotin (Spd-biotin), we developed a new chemo-enzymatic method for probing protein glutathionylation in mammalian cells. Immunoblotting and HPLC analysis indicated that GspS were expressed and functional to produce Gsp in GspS-transfected 293T cells. Spd-biotin was shown to go inside the GspS-transfected 293T cells and subsequently conjugate with endogenous GSH to generate Gsp-biotin. Gsp-biotin S-thiolated proteins were also detected by immunoblotting, suggesting that Gsp-biotin presumably acts like GSH to form mixed disulfide bonds with protein thiols, and that our method is able to effectively label GSH S-thiolated proteins. Meanwhile, we established an efficient procedure to identify GSH S-thiolated proteins. Gsp-biotin S-thiolated proteins were subjected to trypsin digestion, enriched by avidin-based affinity chromatography, hydrolyzed by Gsp amidase to give GSH S-thiolated peptides. Futher liquid chromatography-tandem mass spectrometry analysis led to identification of GSH S-thiolated proteins. Our ultimate goal is to provide an efficient and useful platform to characterize protein S-glutathionylation for large-scale analysis.