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研究生: 黃義澧
Huang, Yi-Li
論文名稱: 利用LC-MS鑑定哺乳類聚醣中差異性鏈結單醣殘基
Identifying specific and differentially linked glycosyl residues in mammalian glycans by targeted LC-MS analysis
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2017
畢業學年度: 105
語文別: 中文
論文頁數: 61
中文關鍵詞: 醣鏈結分析部分甲基化醣醇液相層析串聯式質譜
英文關鍵詞: glycans linkage analysis, partially methylated alditol,, LC-MS
DOI URL: https://doi.org/10.6345/NTNU202202826
論文種類: 學術論文
相關次數: 點閱:87下載:77
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  • 聚醣是由數個單醣藉由糖苷鍵連結而成,由於醣蛋白中的聚醣結構具有高度複雜性,因此了解聚醣中單糖的組成與鍵結就成為重要的工作。在本實驗中開發一個方法,將聚醣經過一連串反應並水解形成 ”部份甲基化糖醇partially O-methylated alditols (PMAs)” 結合液相層析搭配串聯式質譜儀進行分析。實驗流程方面,取得聚醣後將聚醣進行還原,還原端的單醣會被還原而開環,接著進行聚醣的全甲基化,會將聚醣上所有free hydroxyl group的氫置換成甲基(CH3),甲基化聚醣以MALDI-TOF分析確認甲基化反應是否完全,最後進行聚醣的酸水解將聚醣的糖苷鍵破壞形成單醣,此時單醣在醣苷鍵位置會保留OH,最後將所有單醣進行還原得到目標分析物PMA。本實驗分析兩種聚醣的PMA,Fetuin N-glycan與Lewisa所產生的PMA都能經由LC-MS偵測。總而言之,本實驗可以鑑定出聚醣中不同鍵結的單醣,且此方法不須進行額外的衍生化實驗,與LC-MS平台有很好的相容性。

    Glycan is a compound consisting a large number of monosaccharides linked glycosidically. Due to its high complexity of glycan structures on glycoproteins, assessing the configuration and position of glycosidic linkages of a glycan is in great demand. In this study, a method via partially O-methylated alditols (PMAs) from glycan combined with LC-MS analysis is developed. N-glycans were first per-methylated with methyl iodide, and levels of methylation were further confirmed with MALDI-TOF. PMAs were then produced via totally hydrolysis and reduction. PMAs from Fetuin N-glycan and Lewisa were successfully detected by LC-MS analysis. In conclusion, this proposed glycan linkage analysis can be performed without additional derivatization step for GC analysis, and should be suitable for the LC-MS-based platform.

    目錄 目錄 I 圖目錄 IV 表目錄 VI Abstract VII 中文摘要 VIII 縮寫 IX 第一章 緒論 10 第一節 前言 10 第二節 蛋白質的醣基化 10 壹、 聚醣(glycan) 11 貳、 蛋白質醣基化類型 13 參、 蛋白質醣基化功能 16 肆、 醣蛋白分析 17 伍、 單醣的分析 20 陸、 透過PMAA進行聚醣鏈結分析 21 柒、 透過PMA進行聚醣的單醣殘基分析 22 第三節 樣品分析系統介紹 23 壹、 樣品導入系統 (Sample Inlet) 24 貳、 離子源 (Ion Source) 25 參、 質量分析器 (Mass Analyzer) 28 肆、 偵測器 (Detector) 30 伍、 真空系統(Vacuum System) 31 第四節 研究動機與目的 32 第二章 實驗器材與方法 33 第一節 實驗器材 33 第二節 PNGaseF水解Fetuin N-glycan 34 壹、 材料與試劑 34 貳、 實驗步驟 34 第三節 NaBH4 還原N-聚醣(Reduce N-glycan) 35 壹、 材料與試劑 35 貳、 實驗步驟 35 第四節 聚醣的完全甲基化(Per-methylation) 36 壹、 材料與試劑 36 貳、 實驗步驟 36 第五節 C18 spin column純化甲基化聚醣 36 壹、 材料與試劑 36 貳、 實驗步驟 36 第六節 聚醣的酸水解 37 壹、 材料與試劑 37 貳、 實驗步驟 37 第七節 部分甲基化單醣的乙醯化(Acetylation) 37 壹、 材料與試劑 37 貳、 實驗步驟 37 第八節 MALDI-TOF分析甲基化聚醣 38 壹、 分析儀器 38 貳、 基質(Matrix) 38 參、 分析參數 38 肆、 實驗步驟 38 第九節 LC-MS分析部分甲基化單糖 39 壹、 分析儀器 39 貳、 層析參數 39 第三章 結果與討論 41 第一節 MALDI-TOF分析全甲基化醣 41 第二節 LC-MS分析PMAA 42 第三節 LC-MS分析PMA 48 第四節 PMA的相對定量 55 第四章 結論與未來展望 57 第五章 參考文獻 58

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