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研究生： 洪安第
Hung, An-Ti
論文名稱： 邏輯定序引導串聯質譜法應用於乳清蛋白中N-聚醣的結構鑑定
Logically Derived Sequence Tandem Mass Spectrometry for Structural Determination of N-Glycans in Whey Protein
指導教授： 倪其焜
Ni, Chi-Kung
林俊成
Lin, Chun-cheng
口試委員: 吳世雄
Wu, Shih-Hsiung
洪上程
Hung, Shang-Cheng
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2021
畢業學年度： 109
語文別： 中文
論文頁數： 109
中文關鍵詞： N-聚醣 、高效能液相層析法 、質譜分析 、碰撞誘導解離
外文關鍵詞： N-glycan, high-performance liquid chromatography, mass spectrometry, collision-induced dissociation
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    • N-聚醣(N-glycan)具有多種生物學作用並且有時參與病原體相互作用，若想更進一步了解 N-聚醣在生物系統中扮演的角色，鑑定其結構是非常重要的。N-聚醣由數個單醣(Monosaccharides)組成，單醣與單醣間，以醣苷鍵(Glycosidic Bond)鍵結，形成高度複雜的結構。
    到目前為止，已經發展各種不同解醣結構的方法，但仍然沒有一個方法能快速且準確的解出每一個醣的結構。因此，我們開發了一種新的解醣方法，邏輯定序引導串聯質譜法(logically derived sequence, LODES)，利用高效能液相層析法(HighPerformance Liquid Chromatography, HPLC)及質譜分析法(Mass Spectrometry)建立了雙醣的質譜資料庫，並利用醣解離機制的原理，建立解離順序，再藉由碰撞誘導解離(Collision-Induced Dissociation, CID)將寡醣(Oligosaccharides)分解成數個雙醣(Disaccharides)碎片，並將這些雙醣碎片的質譜(Mass Spectra)分別與我們建立的雙醣質譜的資料庫進行比對，藉此快速的鑑定醣的結構，此研究將會利用邏輯定序引導串聯質譜法去鑑定在乳清蛋白中分離出的 N-聚醣。

    N-glycans have multiple biological effects and sometimes participate in pathogen interactions. If we want to know more about the role of N-glycans in biological systems, it is very important to identify its structure.
    By now, many methods have been proposed to identify the structure of oligosaccharides. It is still challenging for these methods to resolve the structures in detail. Therefore, we have developed a new method, logically derived sequence ( LODES). This method was based on the comparison of the disaccharide database with the
    spectra of disaccharides residues dissociated from oligosaccharide by collision-induced dissociation (CID).
    The disaccharides database was built using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS") and the spectra of disaccharide residues produced from CID of oligosaccharides were obtained by ESI-MS".
    In this study, I used LODES to identify the structures of the N-Glycans in whey protein.

    中文摘要-II Abstract-III Contents-IV 1 Introduction-1 1.1 Introduction of Carbohydrates-1 1.2 Introduction of N-glycan-3 1.3 Structure Identification Methods of Carbohydrates-4 2 Experiment Methods-5 2.1 Materials and Reagents-5 2.2 Experimental Section-5 2.2.1 Experimental instrument-5 2.2.2 N-Glycan Release-6 2.2.3 N-Glycan Purification-6 2.2.4 N-glycan Isolation-7 3 Logical Procedure to Determine the Structure of Oligosaccharides-20 3.1 Mechanism of Dehydration-21 3.2 Mechanism of Glycosidic Bond Cleavage-22 3.3 Mechanism of Cross-Ring Dissociation-23 3.4 Logical Procedure for Oligosaccharides-24 4 Result and discussion-25 4.1 Determine the Structure of whey protein N-glycan for complex and hybrid-25 4.1.1 Whey protein N-glycan of m/z=1339-25 4.1.2 Whey protein N-glycan of m/z=1460-34 4.1.3 Whey protein N-glycan of m/z=1501-41 4.1.4.1 Whey protein N-glycan of m/z=1622 for retention time=36.8, 40.8(min.)-46 4.1.4.2 Whey protein N-glycan of m/z=1622 for retention time=39.4, 43.4(min.)-54 4.1.5.1 Whey protein N-glycan of m/z=1663 for retention time=35.6, 39.7(min.)-61 4.1.5.2 Whey protein N-glycan of m/z=1663 for retention time=38.3, 42.3(min.)-67 4.1.5.3 Whey protein N-glycan of m/z=1663 for retention time=39.7, 44.0(min.)-71 4.1.6 Whey protein N-glycan of m/z=1704-73 4.1.7 Whey protein N-glycan of m/z=1746-79 4.2 Determine the Structure of whey protein N-glycan for high-mannose-81 4.2.1 Whey protein N-glycan of m/z=1257-81 4.2.2 Whey protein N-glycan of m/z=1419-87 4.2.3 Whey protein N-glycan of m/z=1581-89 4.2.4 Whey protein N-glycan of m/z=1743-94 4.2.5 Whey protein N-glycan of m/z=1905-99 4.3 Discussion-102 5 Conclusion-106 Reference-107

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