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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65119完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 何國榮(Guor-Rong Her) | |
| dc.contributor.author | Shu-Ting Chen | en |
| dc.contributor.author | 陳淑王亭 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:25:56Z | - |
| dc.date.available | 2022-07-31 | |
| dc.date.copyright | 2012-08-15 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-31 | |
| dc.identifier.citation | chapter1
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65119 | - |
| dc.description.abstract | 本研究利用負離子電灑法二維離子阱質譜儀進行醣蛋白醣鏈及醣胺多醣結構之分析。醣化為蛋白質最主要的轉譯後修飾之一，醣蛋白上的醣鏈在蛋白質的結構與功能上，扮演了重要的角色。對於醣蛋白醣鏈結構之分析，實驗室已開發對胺基苯甲酸乙酯(p-aminobenzoic acid ethyl ester, ABEE)閉環標記醣鏈-負離子電灑法質譜/質譜分析寡醣鍵結點方法，然而對於大於六醣的醣鏈，非還原端的鍵結資訊常相當微弱，難以獲得。因而需於對胺基苯甲酸乙酯閉環標記負離子電灑法質譜/質譜進行分析前，進行鹼逐步水解寡醣反應，始可獲得整個醣鏈的鍵結資訊，然而，此鹼水解過程相當冗長且繁複。為了有效率地獲得醣鏈結構資訊，尋找一個可提供非還原端部分的鍵結資訊的有效方法是必要的，有文獻指出，以質譜/質譜分析含有固定電荷於還原端的醣鏈時，不同於ABEE閉環標記分析，其裂解主要發生自非還原端部分且裂解離子皆包含還原端，因而可反應非還原端部分的鍵結資訊。基於這概念，於醣鏈還原端閉環標記上一個帶有負電荷的衍生試劑，8芘胺-1,3,6-三磺酸(8-aminopyrene-1,3,6-trisulfonate, APTS) 後，再以負離子電灑法質譜/質譜進行分析，以獲得非還原端的鍵結資訊。於醣蛋白醣鏈結構的分析上，本研究提出一個策略為結合ABEE及APTS閉環標記分析醣鏈(大於六醣)結構的鍵結資訊，而可於二次質譜分析時，獲得整個醣鏈的鍵結資訊。
於結合鍵結資訊之策略應用前，先評估APTS閉環標記-負離子電灑法質譜/質譜分析醣鏈鍵結點的方法，結果顯示，此方法可提供非還原端部份之鍵結資訊，且對於非還原端及非還原端以外的鍵結判定所依據的特徵裂解離子不同，因而可區分鍵結點的位置。將APTS閉環標記醣鏈分析鍵結點方法應用於分析直鏈之四醣，結果顯示，於二次質譜分析時，所有鍵結可明確地自非還原端依序被解出。 進一步，將提出的鍵結資訊結合之策略應用到較大的醣蛋白醣鏈結構分析(大於六醣)，以醣蛋白核醣核酸的中性醣鏈為分析物進行策略評估。核醣核酸酶B以醣胜肽酶F進行切醣，藉由ABEE閉環標記醣鏈分析獲得醣鏈還原端部分的鍵結資訊；而藉由APTS閉環標記醣鏈分析獲得醣鏈非還原端部分的鍵結資訊，於二次質譜分析時，結合兩種分析資訊而可獲得中性醣鏈(Man5GlcNAc2、Man6GlcNAc2 、Man8GlcNAc2 、Man9GlcNAc2)的鍵結資訊。 生物體中的醣蛋白，除了含有中性醣鏈(如 : 核醣核酸酶B醣鏈)外，亦含含有更多含唾液酸的複雜型酸性醣鏈，繼之，將結合ABEE及APTS閉環標記分析醣鏈結構之策略運用到醣蛋白酸性醣鏈的結構分析，結果顯示，於APTS閉環標記醣鏈分析時，需先將位於醣鏈尾端的唾液酸進行甲基胺化反應，再以APTS進行閉環標記，以確保整個醣鏈的負電荷係固定在還原端，繼之，以負離子電灑法-質譜/質譜分析，不僅可提供尾端唾液酸的之鍵結資訊 (2-3 及2-6鍵結)，亦可提供靠近非還原端部分的醣鏈鍵結資訊依據APTS閉環標記分析醣鏈方法。此結合鍵結資訊之策略，可成功地鑑定出運鐵蛋白及乳鐵蛋白之雙天線醣鏈，進一步，將此策略運用到運鐵蛋白的三天線醣鏈 (含有二個同質異構醣鏈A3-A及A3-B)，藉由分析ABEE閉環標記三天線醣鏈可獲得二同質異構醣鏈的還原端部分及兩個分支點之鍵結資訊，而藉由快速分離短填充探針搭配自動質譜/質譜分析APTS閉環標記甲基胺化修飾之三天線醣鏈，可分別獲得二個同質異構醣鏈的非還原端部分的鍵結資訊，將兩部分的資訊結合即可獲得二個異構醣鏈的所有鍵結資訊。 醣胺多醣為一長直鏈且被硫酸化修飾的異質多醣體，並參與許多生物體的功能，研究發現，此醣鏈結構(硫酸化型式)會影響醣胺多醣與功能性蛋白質間的交互作用。於醣胺多醣結構分析時，目前，對於大於雙醣的軟骨素之結構分析，要確切地判定每個氮-乙醯半乳醣胺被硫酸化的位置仍有些困難，然而，為了解決此問題，本研究提出了一個策略，藉由位向性選擇切除碳六-氧-硫酸官能基反應搭配負離子電灑法-質譜/質譜分析硫酸軟骨素，比較反應前後分析物的質譜/質譜圖中醣苷鍵裂解離子的偏移情形，以獲得整個軟骨素硫酸化型式的資訊。先以標準品肝素雙醣(heparin disaccharide) 尋找最佳的反應條件。繼之，評估此策略的可行性，以二次質譜分析反應前後的分析物可成功地判定此雙醣的硫酸化型式。進一步，將此分析策略運用到較大的軟骨素分析，以鯊魚軟骨素為分析物，先將其降解成四醣軟骨素後，以胺基液相層析管柱分離多種結構的四醣軟骨素，將各個分離得到的四醣軟骨素收集起來，以提出的醣胺多醣結構判定之策略進行分析。結果顯示，可成功地判定十一種分離得到的四醣軟骨素之硫酸化型式，含有一至三個硫酸根修飾的四醣軟骨素，並以層析峰面積初步地定量十一種結構的四醣軟骨素結構於鯊魚軟骨中所佔的比例。 | zh_TW |
| dc.description.abstract | In this study, negative linear ion trap mass spectrometry was used for structure characterization of oligosaccharides of glycoproteins and glycosaminoglycans. Glycosylation is one of the most abundant post-translational modification of protein. The oligosaccharides of glycoproteins play a critical role in terms of protein function. For structure characterization of oligosaccharide, our earlier study showed that a method based on p-aminobenzoic acid ethyl ester (ABEE) closed-ring labeling and negative ion ESI was presented. For larger oligosaccharides, to obtain linkages near the non-reducing end, a procedure involving alkaline degradation was introduced prior to ABEE labeling. This approach was time-consuming, so an effective method presented to provide linkage information near the non-reducing end is required. Oligosaccharides were labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) before negative MS2 analysis and the fragmentation occurred at the non-reducing end, so linkages near the terminus of oligosaccharides was obtained. The complementary information provided by ABEE and APTS-labeled oligosaccharides was utilized to elucidate the structure of larger oligosaccharides under negative MS2 analysis.
Prior the application of this combined approach, a method based on APTS labeling and negative ion ESI for linkage determination was investigated. The results revealed that the fragmentation of APTS labeling occurred primarily at the terminus. Therefore, linkage information starting at the non-reducing end was provided. In addition, the linkage assignments for terminal and internal linkage were based on different specific linkage fragment ions. Based on these ions, all the linkages of linear oligosaccharides could be determined from the non-reducing to the reducing terminus. Furthermore, the potential of the combined approach was demonstrated using larger neutral oligosaccharides, such as M5G2、M6G2、M8G2、M9G2 cleaved from ribonuclaease B. The results revealed that the linkage and branch assignments could be deduced from complementary information obtained from MS2 spectra of ABEE-labeled and APTS-labeled oligosaccharide. The linkages near the reducing end were derived from the spectrum of ABEE labeling, whereas linkages near the non-reducing end were assigned from the MS2 spectrum of APTS labeling. Many glycoproteins appeared in living organisms contain abundant acidic oligosaccharides decorated with sialic acids. The potential of the combined approach was demonstrated using acidic oligosaccharides cleaved from transferrin and lactoferrin. To assure all negative charges were concentrated at the reducing terminus, a procedure involving methyl amidation was introduced prior to APTS labeling. The results showed that the specific linkage fragments for 2-3 and 2-6 linked sialic acids were obtained and the linkages near the non-reducing end could be determined. This combined approach for linkage assignments was used to deduce the linkages and branches of biantennary oligosaccharides cleaved from transferrin and lactoferrin. In addition, this approach was demonstrated with triantennary oligosaccharides (A3-A and A3-B glycans) cleaved from transferrin. The linkages near the reducing end were obtained from ABEE labeling, whereas linkages near the non-reducing end could be assigned based on a method involving methyl amidation, APTS labeling, and a short C18 packing probe separation coupled with negative ion ESI incorporating data-dependent tandem MS. Consequently, the linkages for A3-A and A3-B could be deduced. For structure characterization of glycosaminoglycans, to successfully deduce the sulfation pattern of glycosaminoglycan, a strategy based on regioselective 6-O-desulfation reaction coupled with negative ESI analysis was developed. The information on sulfation pattern could be obtained based on glycosidic bond cleavages observed in the MS2 spectra of analytes before and after desulfation reaction. This strategy was demonstrated using a standard heparin and a series of tetrasaccharides prepared from shark cartilage chondroitin sulfate D. Additionally, a rougher estimation of the abundances of tetrasaccharides in shark cartilage used direct comparisons of relative peak areas. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:25:56Z (GMT). No. of bitstreams: 1 ntu-101-F95223024-1.pdf: 13175162 bytes, checksum: 458df9ff6478ee58fcb4ee624b2ae3c2 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 摘要 I
Abstract IV 第一章 序論 1 1.1前言 1 1.2. 醣蛋白及蛋白聚醣之質譜分析 4 1.2.1醣蛋白醣鏈結構分析 4 1.2.2蛋白聚醣醣鏈結構分析 8 1.3 電灑法（electrospray,ESI） 11 1.4 離子阱質譜儀 15 1.4.1 三維離子阱 15 1.4.2 二維離子阱（線性離子阱） 21 1.5 研究目的 23 1.6 參考文獻 24 第二章 APTS 閉環標記-負離子電灑法質譜/質譜分析多醣鏈之鍵結點 41 2.1前言 41 2.1.1 醣蛋白的典型醣鏈 43 2.1.2 切醣的方法 45 2.1.3 質譜分析鍵結點之方法 46 2.1.4 研究目的 49 2.2 實驗 51 2.2.1 藥品 51 2.2.2 儀器裝置 52 2.2.3 實驗方法 54 2.3 結果與討論 57 2.3.1 微量寡醣之非還原胺化法衍生化 57 2.3.2 直鏈寡醣閉環標記/質譜/質譜分析鍵結點 59 2.4 結論 64 2.5 參考文獻 65 第三章 APTS 結合 ABEE 閉環標記-負離子電灑法-質譜/質譜分析中性醣鏈之醣結構 89 3.1 前言 89 3.2 實驗 90 3.2.1 藥品 90 3.2.2 儀器裝置 91 3.2.3 實驗方法 93 3.3 結果與討論 97 3.3.1分析M5G2之醣鏈結構 97 3.3.2分析M6G2之醣鏈結構 99 3.3.3 分析M8G2之醣鏈結構 101 3.3.3 分析M9G2之醣鏈結構 104 3.3.5 評估APTS閉環標記分析同分異構醣鏈M7G2結構之可行性 108 3.4 結論 109 3.5 參考文獻 110 第四章 APTS 結合 ABEE 閉環標記-負離子電灑法-質譜/質譜分析複雜型醣鏈之醣鏈結構 131 4.1 前言 131 4.2 實驗 133 4.2.1 藥品 133 4.2.2 儀器裝置 134 4.2.3 實驗方法 138 4.3 結果與討論 143 4.3.1 閉環標記衍生化最佳條件 143 4.3.2 切運鐵蛋白及純化 145 4.3.3 負離子電灑法-質譜/質譜分析APTS閉環標記A2醣鏈 146 4.3.4 唾液酸之羧基修飾 148 4.3.5 區別2-3及2-6鍵結唾液酸醣鏈 150 4.3.6 結合ABEE與APTS分析運鐵蛋白之A1醣鏈 152 4.3.7 結合ABEE與APTS閉環標記分析運鐵蛋白之A2醣鏈 154 4.3.8結合ABEE與APTS閉環標記分析乳鐵蛋白之A1F醣鏈 155 4.3.9結合ABEE與APTS閉環標記分析乳鐵蛋白之A2F醣鏈 156 4.4 結論 164 第五章 負離子電灑法-質譜/質譜分析醣胺多醣之醣鏈結構 203 5.1 前言 203 5.1.1 醣胺多醣的結構 204 5.1.2 質譜分析醣胺多醣之硫酸化型式 208 5.1.3. 研究目的 210 5.2 實驗 211 5.2.1 藥品 211 5.2.2 儀器裝置 212 5.2.3 實驗方法 215 5.3 結果與討論 219 5.3.1 分析策略 219 5.3.2 負離子電灑溶液之探討 221 5.3.3位向性選擇切除碳-六-氧-硫酸官能基反應條件之探討 221 5.3.4 評估分析策略之可行性 227 5.3.5 分析鯊魚軟骨素的硫酸化型式及分佈 228 5.4 結論 243 5.5 參考文獻 244 總結 288 | |
| dc.language.iso | zh-TW | |
| dc.subject | 氮-連接型醣鏈 | zh_TW |
| dc.subject | 鯊魚軟骨素 | zh_TW |
| dc.subject | 醣胺多醣 | zh_TW |
| dc.subject | 鍵結分析 | zh_TW |
| dc.subject | 閉環標記 | zh_TW |
| dc.subject | 對胺基苯甲酸乙酯 | zh_TW |
| dc.subject | 6-三磺酸 | zh_TW |
| dc.subject | 8-芘胺-1 | zh_TW |
| dc.subject | Shark cartilage chondroitin sulfate D | en |
| dc.subject | APTS | en |
| dc.subject | ABE | en |
| dc.subject | Closed-Ring Labeling | en |
| dc.subject | Linkage analysis | en |
| dc.subject | Glycosaminoglycans | en |
| dc.subject | N-linked Oligosaccharides | en |
| dc.title | 電灑法串聯質譜於醣蛋白醣鏈及醣胺多醣醣鏈之研究 | zh_TW |
| dc.title | Structural Characterization of Glycoprotein Oligosaccharides and Glycosaminoglycans by Electrospray Linear Tandem Mass Spectrometry | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 韓肇中(Chau-Chung Han),陳玉如(Yu-Ju Chen),魏國晉(Guor-Jien Wei),賴建成(Chien-Chen Lai),陳逸然(Yet-Ran Chen) | |
| dc.subject.keyword | 氮-連接型醣鏈,8-芘胺-1,3,6-三磺酸,對胺基苯甲酸乙酯,閉環標記,鍵結分析,醣胺多醣,鯊魚軟骨素, | zh_TW |
| dc.subject.keyword | N-linked Oligosaccharides,APTS,ABE,Closed-Ring Labeling,Linkage analysis,Glycosaminoglycans,Shark cartilage chondroitin sulfate D, | en |
| dc.relation.page | 290 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-07-31 | |
| 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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