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標題: | 利用酵素與化學探針偵測蛋白質麩胱甘肽化修飾 Development of a chemo-enzymatic method for probing protein glutathionylation |
作者: | Fu-Tan Hsieh 謝馥檀 |
指導教授: | 林俊宏 |
關鍵字: | 麩胱甘肽,麩胱甘肽,化修飾,麩胱甘肽,-精胺質合成酶,活性氧化分子,蛋白質體, glutathione,glutathionylation,glutathionylspermidine synthetase,reactive oxygen species,proteomics, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 麩胱甘肽化修飾為一種可逆的蛋白質轉譯後修飾,是指麩胱甘肽上的硫醇與蛋白質半胱胺酸的硫醇間以雙硫鍵連接,藉此避免蛋白質上硫醇進行不可逆的氧化反應,此外也能調節蛋白質活性。但現有研究麩胱甘肽化修飾蛋白質之方法仍有缺點與限制。
大腸桿菌中的麩胱甘肽-精胺質合成酶能催化麩胱甘肽與精胺質間醯胺鍵的形成,產生麩胱甘肽-精胺質。本研究利用轉染技術使哺乳類細胞表現麩胱甘肽-精胺質合成酶,並配合 Spd-biotin 探針偵測哺乳類細胞中被麩胱甘肽修飾的蛋白質。透過免疫螢光染色以及高效液相層析儀分析,可在被轉染麩胱甘肽-精胺質合成酶基因的 293T 細胞中觀察到麩胱甘肽-精胺質合成酶蛋白質的表現及麩胱甘肽-精胺質的產生;除此之外,藉由西方墨點法可以確認:Spd-biotin 可進入被轉染麩胱甘肽-精胺質合成酶的 293T 細胞中與內生性的麩胱甘肽連結形成 Gsp-biotin,進而對蛋白質的硫醇基修飾,由實驗結果推論 Gsp-biotin 作用與麩胱甘肽相似能與蛋白質硫醇以雙硫鍵相連。 我們進一步建立有效率的方法鑑定被麩胱甘肽化修飾的蛋白質;前述帶有 Gsp-biotin 修飾的蛋白質,利用胰蛋白酶作用及 streptavidin-膠體進行專一性純化,再以麩胱甘肽-精胺質水解酶將帶有麩胱甘肽的胜肽由膠體上沖堤下來,最後由質譜儀分析,能得到被麩胱甘肽修飾的蛋白質與修飾位置。我們最終的目標為提供一個效率且有用的方法能大規模分析與鑑定麩胱甘肽化之蛋白質。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22183 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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