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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林俊宏(Chun-Hung Lin) | |
| dc.contributor.author | Sheng-Huang Lin | en |
| dc.contributor.author | 林聖皇 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:05:50Z | - |
| dc.date.available | 2017-08-28 | |
| dc.date.copyright | 2012-08-28 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-06 | |
| dc.identifier.citation | 1. Hopkins, F. G. (1921) Biochem J 15, 286-305
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64889 | - |
| dc.description.abstract | 麩胱甘肽 (GSH) 藉由其上的硫醇與蛋白質半胱胺酸 (cysteine) 的硫醇形成雙硫鍵稱為蛋白質的麩胱甘肽化 (S-glutathionylation) 。 S-glutathionylation 是一種可逆的蛋白質轉譯後修飾反應，能夠調控細胞內氧化還原平衡與保護細胞避免過度氧化。雖然目前有許多的方法可以偵測蛋白質的S-glutathionylation，但大部份的方法中並無法直接地獲得特定修飾位置的資訊。在本實驗室之前的研究中提出了利用將大腸桿菌的 Gsp synthetase 與 biotinyl-spermine 送入人類細胞中，以鑑定細胞內含有 S-glutathionylation 的蛋白質及其修飾位置。這個方法提供了有效率且可大規模分析相關的蛋白質體學。在本論文中，我們應用並最佳化此以 Gsp-biotin 為基礎的親合性純化修飾蛋白質，另外結合質譜分析，達到專一性的辨識 GSH 修飾的位置，以及對被 S-glutathionylation 的蛋白質進行定量。
在本論文中，我們使用蛋白質酪胺酸磷酸水解酶1B (protein tyrosine phosphatase 1B, PTP1B) 作為模式蛋白，以探討在蛋白質和胜肽層級上胰蛋白酶水解以及純化 GSH 修飾的蛋白質/胜肽的效率，建立及最佳化整個純化以及樣品前處理的步驟。目前為止，利用質譜分析 GSH-PTP1B 的極限最低可達 62 ng 。我們也使用 MS1 細胞株，一個在探討氧化還原研究中被廣泛使用的細胞株，來探討所建立的純化 GSH 修飾蛋白質/胜肽步驟的效率。於細胞外的條件下對 MS-1 細胞蛋白質進行 GSH 的修飾並結合質譜分析，目前辨識到了472條含有 GSH 修飾的胜肽以及其所屬的317個蛋白質。於辨識到的蛋白質中，其中有一些已有文獻廣泛的探討它們的 S-glutathionylaiton 。此外，在本研究所開發的方法中，不但可以專一性的辨識胜肽鏈上被 GSH 修飾的位置，還可以分辨同一條胜肽鏈上具有多個 cysteine ，哪一個 cysteine 是被 GSH 所修飾。 在免標定定量法的部分，我們結合高敏感性的質譜分析以及生物資訊學的工具 (IDEAL-Q) ，藉由估計萃取的離子層析圖譜 (extracted ion chromatography, XIC) 訊號峰底下的面積以估算各 S-glutathionylation 胜肽的量。我們在 MS-1 細胞的蛋白質中加入一序列不同量的 GSH-PTP1B ，在 LC-MS/MS 分析之後計算其被 S-glutathionylation 的三條胜肽鏈 (包括 HEASDFPC32(GSH)R, GSLKC121(GSH)AQYWPQK, ESGSLSPEHGPVVVHC215(GSH)SAGIGR ) 訊號峰下的面積。並以面積對濃度作圖以得到標準曲線，獲得定量的範圍落在 1.8 pmol 到 3.9 nmol 之間。 最後，在結合此一整套純化以及質譜分析的流程以及定量的方法之後，可以進一步探討 S-nitrosylation 與 S-glutathionylation 的關係，以及 S-nitrosylation 的穩定性與反應性。本論文不但提供一個有用的方法鑑定 S-glutathionylation 的蛋白質體，並可以深入探討細胞內氧化還原的調控機制。 | zh_TW |
| dc.description.abstract | Glutathione (GSH) forms a disulfide bond with cysteines of proteins, a dynamic way to regulate cellular redox balance and protect cells from oxidative damage. Although several methods are available to detect glutathionylated proteins, few of them provide direct assignment of modified cysteine residues. Previously we demonstrated an efficient method suitable for large-scale characterizations of glutathionylated proteins in vivo by introducing E.coli glutathionylspermindine synthetase (GspS) and biotinyl-spermine to human cells. In this thesis, we applied the method and optimized the conditions for the enrichment and subsequent mass spectrometric analysis to achieve site-specific GSH identification and quantitative proteomics. We utilized protein tyrosine phosphatase 1B (PTP1B), a well-known and extensively studied protein in redox biology, as a model to optimize the procedures of trypsin digestion and enrichment on both the protein and peptide levels. The detection limit of GSH-PTP1B was found to be 1.8 pmol. In MS-1 endothelial cells we have identified 472 unique glutathionylated peptides (mascot score ≥26, false decoy rate <2.21%) resulting from 317 glutathionylated proteins. Some of which were known to be glutathionylated. In addition, we are able to distinguish between S-glutathionylation and other cysteine modifications in one peptide. The label-free quantitative approach was carried out by integration of highly sensitive mass spectrometric techniques with bioinformatics tools (IDEAL-Q) to calculate the extracted ion chromatogram (XIC) of glutathionylation level on peptides. That is to say, we spiked a series of quantified PTP1B into MS-1 cell lysate and calculated the peak areas of three PTP1B peptides (including HEASDFPC32R, GSLKC121AQYWPQK, ESGSLSPEHGPVVVHC215SAGIGR ) during LC-MS/MS analyses. The range of quantitation is found between 1.8 pmol and 3.9 nmol. This developed method will be useful not only to identify and characterize glutathionylated proteins, but also to decipher how these identified cysteines are subjected to redox regulation. | en |
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| dc.description.tableofcontents | 謝 誌 I
縮寫表 II 中文摘要 IV Abstract VI 第一章 緒論 1 1.1 Glutathione 簡介 1 1.2 Glutathionylation 簡介 3 1.2.1 細胞內的硫醇與氧化還原狀態 3 1.2.2 Glutathionylation 的形成機制 4 1.3 簡介當前研究蛋白質 S-glutathionylation 的方法 7 1.3.1 以放射性同位素 35S 標定 GSH 7 1.3.2 利用 biotin 標定的 GSH 及其類似物研究蛋白質的 S-glutathionylation 8 1.3.3 利用 glutathione 的抗體進行蛋白質 S-glutathionylation 的分析 9 1.3.4 Glutaredoxin (Grx) 結合 biotin switch 法 9 1.3.5 Glutathione S-transferase (GST) 偵測法 10 1.3.6 GSH 或 GSH analogs 固定偵測法 11 1.3.7 總結現有方法之限制 11 1.4 Glutathionylspermidine synthetase/amidase (GspSA) 的簡介 11 1.5 常見結合液相層析-質譜分析 (liquid chromatography mass spectrometry) 的定量方法 12 1.5.1 穩定同位素標定定量法 (Stable isotope labeling quantiation) 13 1.5.1.1 Stable isotope labeling with amino acids in cell culture (SILAC) 13 1.5.1.2 Isotpe-coded affinity tag (ICAT) 14 1.5.1.3 Isobaric tags for relative and absolute quantitation (iTRAQ) 與 Tandem mass tags (TMT) 15 1.5.2 免標定定量法 (Label-free quantitation) 16 1.6 蛋白質的 S-nitrosylation 簡介 17 1.7 實驗動機 17 1.8 實驗流程圖 19 第二章 材料與方法 20 2.1 實驗藥品 20 2.2 表現質體 (Plasmid) 與菌種 20 2.2.1 表現質體與基因 20 2.2.2 菌種 20 2.3 細胞培養 20 2.3.1 細胞種類 20 2.3.2 培養液配製 21 2.3.3 繼代培養 21 2.4 利用化學酵素 (chemoenzymatic) 合成法合成與純化 Gsp-biotin 之化合物 21 2.4.1 Gsp-biotin 小分子的合成 21 2.4.2 ATP回復系統 (ATP regenerating system) 23 2.4.3 薄層層析法 (Thin layer chromatography, TLC) 23 2.4.4 Gsp-biotin 小分子的純化 24 2.4.5 Gsp-biotin 定性分析 24 2.4.5.1 以質譜儀鑑定其純度與分子量 24 2.4.5.2 以 Gsp amidase 對 Gsp-biotin 小分子進行定性分析 24 2.5 以快速蛋白層析儀 ( Fast protein liquid chromatography, FPLC ) 純化Gsp-biotin 小分子以及 His-tagged 蛋白質 25 2.5.1 利用陽離子交換樹脂管柱純化 Gsp-biotin 小分子 25 2.5.2 使用 Hitrap HP Chelating 管柱純化 His-tagged 蛋白質 25 2.6 蛋白質定量法 26 2.6.1 Bradford 定量法 26 2.6.2 BCA定量法 27 2.7 蛋白質膠體電泳分析 (Polyacryamide Gel Electrophoresis) 28 2.8 西方點墨法 (Western blot) 29 2.9 SDS PAGE染色法 30 2.9.1 Coomassie Blue染色法 30 2.9.2 銀染法 (Silver stain) 31 2.10 建立in vitro下純化Gsp-biotin S-thiolated 蛋白質或胜肽的方法 32 2.10.1 細胞裂解 (Cell lysis) 32 2.10.2 以Gsp-biotin在in vitro下對蛋白質進行標定 32 2.10.3 以 iodoacetamide (IAM) 對蛋白質 cysteine 上自由的硫醇進行遮蔽 33 2.10.4 去除過量 Gsp-biotin 的方法 33 2.10.4.1 分子量大小篩選管柱法 (Size exclusion column) 33 2.10.4.2 透析法 (Dialysis) 33 2.10.4.3 丙酮沉澱法 (Acetone precipitation) 33 2.10.5 以胰蛋白酶 (Trypsin) 將蛋白質切成胜肽 34 2.10.5.1 在尿素 (Urea) 作用下進行胰蛋白酶剪切 34 2.10.5.2 在三氟乙醇 (Trifluoroethanol) 作用下進行胰蛋白酶剪切 34 2.10.6 以 Streptavidin agarose bead 純化Gsp-biotin修飾的胜肽 35 2.10.7 利用Gsp amidase將純化出之胜肽上的Gsp-biotin水解成GSH 35 2.11 質譜分析 36 2.11.1 去鹽 (Desalting) 36 2.11.2 MALDI-TOF 分析 36 2.11.3 LC-ESI-MS/MS 分析 36 2.11.4 Collision energy 37 2.11.5 資料庫搜尋與數據分析 38 2.12 結合質譜技術之免標定定量法定量分析 39 2.12.1 質譜定量範圍分析 39 2.12.2 以免標定定量法進行質譜定量分析 39 2.13 細胞內蛋白質的亞硝基化反應 (nitrosylation) 40 第三章 實驗結果 41 Part I 在in vitro下合成與純化Gsp-biotin小分子的方法之建立 41 3.1.1 Gsp-biotin小分子的酵素合成法 41 3.1.2 Gsp-biotin小分子的純化 41 3.1.3 Gsp-biotin小分子的定性分析 42 Part II Gsp-biotin修飾之蛋白質/胜肽於質譜分析前之樣品處理方法 44 3.2.1 在in vitro下製備Gsp-biotin修飾的PTP1B 蛋白質 45 3.2.2 移除未反應的Gsp-biotin 條件測試 46 3.2.3 胰蛋白酶水解 (Trypsin digestion) 條件選擇 48 3.2.4 以streptavidin純化Gsp-biotin修飾蛋白質/胜肽的沖提 (elution) 條件之選擇 50 Part III 以質譜分析 Gsp-biotin 修飾胜肽序列特性 52 3.3.1 Gsp-biotin 修飾 PTP1B 的質譜分析 52 3.3.2 ESI-Q-TOF的裂片 (fragmentation) 能量選擇 55 3.3.3 以質譜分析 MS-1 內皮細胞中 in vitro S-glutathionylation 潛在蛋白質 58 Part IV 以質譜為基礎之免標定定量法之建立 60 3.4 以PTP1B作為免標定定量法 (label-free quantitation) 的偵測動態範圍(dynamic range) 分析 60 Part V 利用Gsp-biotin探討S-glutathionylation與S-nitrosylation的關係 64 3.5.1 Cysteine的nitrosylation可能會促進glutathionylation的發生 64 3.5.2 以Gsp-biotin 在 in vitro 下探討 S-nitrosylated cysteine (nitrosothiol) 的穩定性 (stability) 以及反應性 (reactivity) 65 第四章 討論 69 4.1 Gsp-biotin 小分子的合成原理與 ATP 回復系統 69 4.2 移除未修飾的Gsp-biotin 方法之探討 70 4.3 在TFE條件下可以幫助胰蛋白酶水解蛋白質 72 4.3 Gsp amidase無法有效地將受到修飾的蛋白質/胜肽從streptavidin上沖提下來 75 4.4 Cysteine 上硫醇基 S-nitrosylation 的穩定性與反應性 75 第五章 結論與未來展望 81 第六章 參考文獻 82 附錄一 In vitro 下Gsp-biotin 修飾蛋白/胜肽純化流程 91 附錄二 質譜鑑定 GSH 修飾胜肽理論分子量對照表 92 附錄三 Bio-rad protein assay kit (Cat. 500-0006) 物質相容性表 93 | |
| dc.language.iso | zh-TW | |
| dc.subject | 麩胱甘肽 | zh_TW |
| dc.subject | 化 | zh_TW |
| dc.subject | Gsp-biotin | zh_TW |
| dc.subject | 蛋白質體 | zh_TW |
| dc.subject | 純化 | zh_TW |
| dc.subject | 專一性的辨識 | zh_TW |
| dc.subject | 免標定定量法 | zh_TW |
| dc.subject | label-free quantitation | en |
| dc.subject | S-glutathionylation | en |
| dc.subject | Gsp-biotin | en |
| dc.subject | proteomics | en |
| dc.subject | enrichment | en |
| dc.subject | site-specific identification | en |
| dc.title | 建立蛋白質麩胱甘肽化特定位鑑定方法與結合質譜的免標定定量法策略 | zh_TW |
| dc.title | Site-specific Identification and Label Free Quantitation of S-glutathionylated Proteins | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳玉如(Yu-Ju Chen),林俊成(Chun-Cheng Lin) | |
| dc.subject.keyword | 麩胱甘肽,化,Gsp-biotin,蛋白質體,純化,專一性的辨識,免標定定量法, | zh_TW |
| dc.subject.keyword | S-glutathionylation,Gsp-biotin,proteomics,enrichment,site-specific identification,label-free quantitation, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-07 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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