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研究生：	林家宏 Chia-Hung Lin
論文名稱：	利用聚精胺酸修飾之奈米鑽石濃縮萃取磺酸化胜肽與肝素 Using Polyarginine-coated Nanodiamonds to Enrich and Extract Sulfopeptides and Heparin
指導教授：	林震煌 Lin, Cheng-Huang 張煥正 Chang, Huan-Cheng 吳志哲 Wu, Chih-Che
學位類別：	碩士 Master
系所名稱：	化學系 Department of Chemistry
論文出版年：	2011
畢業學年度：	99
語文別：	中文
論文頁數：	57
中文關鍵詞：	奈米鑽石、磺酸化、親和性層析、基質輔助雷射脫附游離飛行時間質譜儀、肝素
英文關鍵詞：	nanodiamond, sulfonation, affinity chromatography, MALDI-TOF MS, heparin
論文種類：	學術論文
相關次數：	點閱：118 下載：10
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磺酸化與磷酸化反應是控制細胞內蛋白質功能重要的後轉譯修飾 (Post-Translational Modification，PTM)，在醣類結構上也是相當常見。細胞表面的蛋白聚醣 (Proteoglycan) 銜接出不同硫酸化雙醣為單位的糖胺多醣 (Glycosaminoglycans，GAGs)，例如肝素或硫酸乙醯肝素 (heparin/heparan sulfate) 影響著多種生物反應，包含抗凝血功能、細胞的生長、調控細胞離子濃度、癌症還有細菌或病毒感染，甚至與遺傳疾病都息息相關。基質輔助雷射脫附游離飛行時間質譜儀 (MALDI-TOF MS)是用來分析蛋白質與醣類結構的工具之一，然而後轉譯修飾的醣類或胜肽在生物中含量甚少，所以利用質譜分析之前，樣品的濃縮與純化則是不可或缺的。本篇利用聚精胺酸修飾之奈米鑽石對磺酸化和磷酸化兩種後轉譯修飾胜肽親和性比較，發現磺酸化胜肽具有優先親和性。利用此技術，也能從高含量蔗糖溶液中選擇性萃取微量肝素雙醣。另外還發現於3-aminoquinoline (3-AQ) 基質中添加氨化物1,1,3,3-tetramethylguanidine (TMG) 不只能夠降低肝素雙醣於質譜分析下的磺酸根裂解，還能增強去質子離子的訊號。此奈米鑽石固相萃取技術結合質譜分析硫酸醣類，在未來將有利於了解細胞膜表面發生之病毒感染。

Sulfonation and phosphorylation are important post-translational modifications (PTMs) of protein function in cells and occur frequently in oligosaccharides. The glycosaminoglycans (GAGs), which are characterized by a variably sulfated repeating disaccharide unit, bind with cell surface proteoglycans. For example, heparin/haparan sulfates influence numerous biological processes which include anticoagulation, cellular physiology, ionic strength regulation, cancer, viral invasion, bacteria invasion and genetic diseases. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the tools for the structure analysis of proteins and oligosaccharides. However, the low abundance and low stoichiometry of post-translationally modified oligosaccharides and peptides in organisms make isolation and concentration of the compound indispensable prior to MS analysis. In this study, we utilize polyarginine-coated nanodiamond as a high affinity nanoprobe for sulfopeptides and phosphopeptides. We compared the affinity of there two post-translationally modified peptides toward the nanoparticle and found preferential adsorption of sulfopeptide in their mixture. With this technique, we are able to selectively extract heparin disaccharides in high abundant sucrose solution. Additionally, we found that adding 1,1,3,3-tetramethylguanidine (TMG) to 3-aminoquanoline (3-AQ) matrix not only reduces the sulfate fragmentation of heparin disaccharides but also enhances the signal of the protonated ions of MALDI-TOF MS analysis. We conclude that MALDI MS combined with this nanodiamond–based solid phase extraction is a useful technique. It can facilitate our understanding of viral invasion through interaction with sulfate saccharides on cell membrane surface in the future.

Abstract
摘要
緒論..............................................................................................................................................7
1 前言.....................................................................................................................................7
2 蛋白質的磺酸化後轉譯修飾...............................................................................................7
3 葡萄醣胺聚醣.......................................................................................................................9
4 磺酸化分子與肝素寡醣類的分析鑑定方法.....................................................................13
4.1 同位素標記法...........................................................................................................13
4.2 電泳法.......................................................................................................................14
4.3 HPLC (High-performance liquid chromatography) ...................................................15
4.4 MALDI-TOF MS (Matrix Assisted Laser desorption ionization Time-of-flight Mass spectrometry)................................................................................................................................16
4.5 基質添加物...............................................................................................................17
5 胍基團與磺酸基團之間的非共價鍵結.............................................................................19
6 奈米鑽石做為固相萃取（solid-phase extraction）的材料..............................................23
7 實驗目的與應用.................................................................................................................24
實驗............................................................................................................................................25
1 藥品與材料.........................................................................................................................25
2 儀器設備.............................................................................................................................26
3 實驗步驟.............................................................................................................................27
3.1 奈米鑽石製備...........................................................................................................27
3.2 聚精胺酸的修飾.......................................................................................................28
3.3 微波輔助酵素消化蛋白質........................................................................................29
3.4 基質溶液的配置.......................................................................................................30
3.5 奈米鑽石萃取與磷酸化胜肽混合的胰島素鏈A....................................................30
3.6 複雜環境下奈米鑽石萃取肝素雙醣........................................................................31
3.7 聚精胺酸奈米鑽石對肝素雙醣有效吸附量的估計................................................33
結果與討論................................................................................................................................34
1 奈米鑽石的粒徑分佈(size distribution) 與界達電位(zeta potential) 的量測.............34
2 聚精胺酸奈米鑽石對胰島素鏈A的萃取........................................................................35
2.1 聚精胺酸奈米鑽石對胰島素鏈A有效吸附量的估計...........................................35
2.2 聚精胺酸奈米鑽石對磺酸化胜肽與磷酸化胜肽的親和性比較............................36
3 聚精胺酸奈米鑽石對肝素雙醣的萃取.............................................................................39
3.1 一般MALDI基質於肝素雙醣的分析.....................................................................39
3.2 離子液體基質(Ionic Liquid Matrix，ILM)於肝素雙醣的分析..............................42
3.3 基質3-AQ與基質添加物TMG於肝素雙醣的分析..............................................44
3.4 聚精胺酸奈米鑽石對肝素雙醣有效吸附量的估計................................................46
3.5 聚精胺酸奈米鑽石於高濃度蔗糖環境下對肝素雙醣的萃取................................47
3.6 聚精胺酸奈米鑽石肝素雙醣的萃取偵測極限........................................................48
3.7 聚精胺酸奈米鑽石對人工合成之硫酸化雙醣萃取................................................50
結論..................................................................................................................................53
參考資料..........................................................................................................................54
附錄...............................................57
                                

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