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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 何國榮(Guor-Rong Her) | |
| dc.contributor.author | Chih-Yu Lin | en |
| dc.contributor.author | 林芝毓 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:13:20Z | - |
| dc.date.available | 2015-08-16 | |
| dc.date.copyright | 2010-08-16 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47698 | - |
| dc.description.abstract | 醣蛋白之表現會隨著生理狀態和疾病型式而改變，並與疾病發展相關，因此研究不同狀態下醣蛋白之變化，對於疾病之診斷可提供有用的資訊。醣蛋白濃度、醣鏈分佈及醣化程度皆可能影響醣蛋白之表現，因此本研究主要目的在於提出一分析的策略可用於評估不同狀態下，醣蛋白濃度、醣鏈分佈和醣化程度的相對變化量。藉由比較醣胜肽和胜肽在不同狀態時的質譜訊號相對強度，以獲得醣蛋白濃度、醣化程度、醣鏈分佈之相對變化。利用d0-甲醛與d2-甲醛同位素標記兩不同狀態的醣胜肽，以纖維素微晶體純化醣胜肽，並以電灑法質譜儀偵測以逹到相對定量之分析。為了證實策略之可行性，首先以核糖核酸酶B作為探討對象，探討新策略在以下不同狀態之可行性：(1).醣蛋白濃度改變、醣鏈分佈和醣化程度皆不變；(2).醣鏈分佈改變、醣蛋白濃度與醣化程度皆不變；(3).醣化程度改變、醣蛋白濃度與醣鏈分佈皆不變；(4).醣蛋白濃度、醣鏈分佈、醣化程度均改變。證實所提出之相對定量策略方法可有效地區辨不同狀態下醣蛋白表現之變化，來自於醣蛋白濃度改變、醣化程度改變或醣鏈分佈改變之貢獻分別為何。此外，利用電灑法離子阱質譜分析醣胜肽，藉由離子阱質譜儀具有多次質譜/質譜之能力，於正離子模式下，以質譜/質譜(MS2)獲得醣鏈組成資訊，並以質譜/質譜/質譜(MS3)獲得醣化位置之資訊；於負離子模式下，以多次質譜/質譜(MSn)方式可獲得醣鏈鍵結資訊。
進一步測試策略於複雜樣品具有一個以上醣化位置且醣鏈含有唾液酸之醣蛋白之適用性，選用乳鐵蛋白作為分析目標，以逆相液相毛細管液相層析取代纖維素微晶體純化步驟。實驗結果證實策略於複雜樣品可獲得醣蛋白濃度、醣鏈分佈和醣化程度的相對變化量。 將分析策略應用於真實樣品，選擇母乳中的乳鐵蛋白為真實樣品之例子。利用乳鐵蛋白標準品及市面上購得之鮮乳評估純化方法的可行性，且將各個純化步驟做最佳化的探討，再將收集得到之不同哺乳階段母乳樣品進行純化。最後將分析策略應用於不同哺乳期間母乳中的乳鐵蛋白，成功區別醣蛋白表現之變化來自醣蛋白濃度變化、醣化程度變化、或醣鏈分佈變化之貢獻為何。 | zh_TW |
| dc.description.abstract | Glycoproteins are vared with regulatory states, type of disease, and even with disease progression. Therefore, comparative studies of the change of glycoproteins may provide useful information for the diagnosis of disease. Several causes including the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile may account for the difference if the signal of a glycopeptide was found to be changed. A new strategy was proposed for comparative analyses of glycoprotein in which the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be differentiated. Comparative analysis was performed by stable isotope labeling using formaldehyde-d0 and formaldehyde-d2 along with cellulose microcrystalline enrichment and mass spectrometry analysis. The utility of the proposed strategy was demonstrated with ribonuclease B. The feasibility of the new strategy under different situations was studied as following: (1)glycoprotein concentration changed, glycan profile and glycosylation site occupancy fixed; (2)glycan profile changed, glycoprotein concentration and glycosylation site occupancy fixed; (3)glycosylation site occupancy changed, glycoprotein concentration and glycan profile fixed; (4) glycoprotein concentration, glycan profile and glycosylation site occupancy all changed. As a consequence, the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be obtained simultaneously. ESI-MS analysis of glycopeptides was performed on a linear ion trap mass spectrometer. With the capability of MS2 and MS3 of the instrument, the site of glycosylation and the glycan sequence may also be obtained in positive-ion mode. The negative-ion mode of MSn was useful for glycan structural analysis.
To test the feasibility of the proposed strategy in complex glcoprotein with more than one glycosylation site and sialylated N-glycans, lactoferrin was chosen as the model compound. Cellulose microcrystalline enrichment was replaced by reversed-phase liquid chromatography. The results showed that the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be obtained in complex glycoprotein sample. To apply the strategy on lactoferrin from human milk of different stages of lactation period, first optimize purification method by lactoferrin standard and market milk. Lactoferrin was successfully purified form human milk. The glycoprotein change of lactoferrin from different lactation stages due to glycoprotein concentration, glycosylation site occupancy and glycan profile could be differentiated by the strategy. | en |
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| dc.description.tableofcontents | 中文摘要 i
英文摘要 iii 圖目錄 7 表目錄 19 第一章 序論 1.1 前言 21 1.2. 醣蛋白之質譜分析 25 1.2.1醣蛋白定量分析 25 1.2.2醣蛋白上醣化之分析 27 1.3 電灑法（electrospray,ESI） 30 1.4 離子阱質譜儀 34 1.4.1 三維離子阱 34 1.4.1.1 離子阱之構造 34 1.4.1.2 離子阱之分析電壓 34 1.4.1.3 離子阱的分析步驟 37 1.4.1.4 大氣壓界面與離子阱配合設計 39 1.4.2 二維離子阱（線性離子阱） 39 1.5 研究目的 41 1.6 參考文獻 43 第二章 醣蛋白醣化相對定量之策略建立與評估 2.1前言 71 2.2 實驗 76 2.2.1虊品 76 2.2.2 儀器裝置 77 2.2.2.1質譜儀 77 2.2.2.2高速離心機 77 2.2.2.3吹氣濃縮儀 77 2.2.2.4加熱恆溫反應器 78 2.2.3 實驗方法 79 2.2.3.1利用α-Mannosidase酵素修飾醣蛋白之醣鏈 79 2.2.3.2 醣蛋白之水解消化 79 2.2.3.3 還原甲基化標記反應 80 2.2.3.4 纖維素微晶體純化醣胜肽 80 2.2.3.5 ZipTipC18淨化 (Clean-up)胜肽 81 2.2.3.6 質譜儀分析 81 2.2.3.7 Sequest蛋白質資料庫鑑定 82 2.3 結果與討論 83 2.3.1 分析策略 83 2.3.2 還原甲基化法標記醣胜肽 89 2.3.3 再現性之評估 91 2.3.4 醣胜肽之相對定量 93 2.3.5 蛋白質濃度(glycoprotein concentration)之相對定量 94 2.3.6 醣鏈分佈(glycan profile)改變之相對定量 96 2.3.7 醣化程度(glycosylation site occupancy)之相對變化 99 2.3.8 醣蛋白濃度改變、醣化程度改變、醣鏈分佈改變之相對定量 101 2.3.9醣化位置之鑑定與醣鏈結構之偵測 103 2.3.9.1醣鏈序列和醣化位置之鑑定 103 2.3.9.2 醣鏈分佈(glycosylation profile)之偵測 107 2.3.9.3 醣鏈鍵結(glycan linkage)之鑑定 108 2.4 結論 111 2.5 參考文獻 113 第三章 毛細液相層析電灑法二維離子阱質譜於乳鐵蛋白醣化相對定量之研究 3.1 前言 161 3.2 實驗 162 3.2.1藥品 162 3.2.2 儀器裝置 163 3.2.2.1 逆相毛細管液相層析 163 3.2.2.2質譜儀 166 3.2.2.3高速離心機 166 3.2.2.4吹氣濃縮儀 166 3.2.2.5加熱恆溫反應器 167 3.2.3 實驗方法 168 3.2.3.1 醣蛋白之水解消化 ( Glycoprotein Digestion ) 168 3.2.3.2 利用還原甲基化法於醣胜肽氮端標記 168 3.2.3.3 ZipTipC18淨化胜肽 169 3.2.3.4質譜儀分析 169 3.2.3.5 逆相毛細管液相層析 169 3.2.3.6 蛋白質資料庫鑑定 170 3.3 結果與討論 171 3.3.1 分析策略 171 3.3.2 逆相毛細管液相層析質譜之建立及分離效能之評估 174 3.3.3 還原甲基化法標記醣胜肽反應條件之探討 177 3.3.4 同位素標記醣胜肽和胜肽於逆相毛細管液相層析之同位素效應探討 182 3.3.5定量模式 184 3.3.6 醣胜肽之相對定量 186 3.3.6.1醣化位置Asn 479 186 3.3.6.2醣化位置Asn 138 188 3.3.7 醣蛋白濃度改變之相對定量 189 3.3.8蛋白質濃度變化、醣鏈分佈變化、醣化程度變化之探討 191 3.4 結論 195 3.5 參考資料 197 第四章 應用於初乳中乳鐵蛋白醣蛋白之研究 4.1 前言 249 4.2 實驗 251 4.2.1 虊品 251 4.2.2儀器裝置 252 4.2.2.1逆相毛細管液相層析 252 4.2.2.2質譜儀 252 4.2.2.3高速離心機 252 4.2.2.4吹氣濃縮儀 252 4.2.2.5加熱恆溫反應器 252 4.2.2.6 硫酸十二酯鈉聚丙烯醯胺凝膠電泳 252 4.2.3實驗方法 253 4.2.3.1純化母乳中的乳鐵蛋白 253 4.2.3.2硫酸十二酯鈉聚丙烯醯胺凝膠電泳 254 4.2.3.3醣蛋白水解消化 257 4.2.3.4利用還原甲基化法於醣胜肽氮端標記 258 4.2.3.5 ZipTipC18淨化胜肽 258 4.2.3.6 利用刀豆凝集素A(Con A lectin)純化醣胜肽 258 4.2.3.7 醣胜肽去醣化反應 259 4.2.3.8質譜儀分析 259 4.2.3.9 逆相毛細管液相層析 259 4.2.3.10 MASCOT蛋白質資料庫鑑定 259 4.3 結果與討論 260 4.3.1純化母乳中乳鐵蛋白之流程設計 260 4.3.1.1硫酸銨沈澱法做初步純化 260 4.3.1.2 利用刀豆凝集素A純化醣蛋白 263 4.3.1.3 利用鮮奶測試純化流程之結果 264 4.3.1.4 純化不同哺乳階段母乳中的乳鐵蛋白之結果 265 4.3.2 母乳純化之乳鐵蛋白於溶液中水解之可行性評估 267 4.3.3相對定量分析策略應用於母乳中乳鐵蛋白分析 270 4.3.3.1 由策略獲得蛋白質濃度、醣鏈分佈及醣化程度變化 270 4.3.3.2 由去醣化醣胜肽獲得醣化程度以驗證策略之可行性 272 4.4 結論 276 4.5 參考文獻 277 總結 311 | |
| dc.language.iso | zh-TW | |
| dc.subject | 質譜 | zh_TW |
| dc.subject | 醣蛋白 | zh_TW |
| dc.subject | 蛋白質濃度 | zh_TW |
| dc.subject | 醣化程度 | zh_TW |
| dc.subject | 醣鏈分佈 | zh_TW |
| dc.subject | glycan profile | en |
| dc.subject | protein concentration | en |
| dc.subject | glycoprotein | en |
| dc.subject | mass spectrometry | en |
| dc.subject | glycosylation site occupancy | en |
| dc.title | 同位素標記電灑法質譜於不同狀態下醣蛋白變化之研究 | zh_TW |
| dc.title | Comparative Studies of Glycoprotein Using Isotope Labeling and Electrospray Ion Trap Mass Spectrometry | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 李茂榮,凌永健,賴建成,陳逸然 | |
| dc.subject.keyword | 醣蛋白,蛋白質濃度,醣化程度,醣鏈分佈,質譜, | zh_TW |
| dc.subject.keyword | glycoprotein,protein concentration,glycosylation site occupancy,glycan profile,mass spectrometry, | en |
| dc.relation.page | 312 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-13 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| 顯示於系所單位： | 化學系 | |
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