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標題: | 毛細電泳質譜分析高甘露醣鏈及其鍵結異構物之研究 CE-MS Analysis of High Mannose Glycans and Isomers |
作者: | En-Tzu Lu 陸恩慈 |
指導教授: | 何國榮 |
關鍵字: | 毛細電泳質譜,高甘露醣鏈,8-芘胺-1,3,6-三磺酸,硼酸緩衝溶液,聚氧化乙烯,雙槽式電泳質譜介面,鍵結異構物, CE-MS,high-mannose glycans,APTS,borate buffer,PEO,double junction interface,isomers, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 由於醣鏈結構相當複雜,在質譜分析之前分離醣鏈是必須的。在本研究中使用毛細電泳銜接質譜,分析核醣核酸酶B的高甘露醣鏈;以閉環和開環衍生的方法,將醣鏈分別標記對胺基苯甲酸(ABA)和8-芘胺-1,3,6-三磺酸(APTS),並探討最佳分離條件。毛細電泳分離方法可分為兩個系統,一個是毛細區帶電泳,以硼酸緩衝溶液作為分離條件,於高pH值、正向模式電場下分析; 另一個系統為毛細膠體電泳,以添加聚氧化乙烯(polyethylene oxide, PEO)膠體分子的醋酸銨溶液作為分離條件,於低pH值、反向模式電場下分析。於UV偵測下,相較於ABA標記和APTS閉環標記,以APTS開環標記醣鏈作為分離對象於毛細區帶電泳和毛細膠體電泳都有較佳分離結果。將兩個分離系統銜接質譜時,分離條件中的硼酸會抑制分析物訊號,而膠體分子PEO則會汙染質譜的離子源,為此使用實驗室所開發的雙槽式介面(double junction interface)銜接電泳與質譜;此介面包含三部分:液體接合槽、銜接管及低鞘流介面。在硼酸緩衝溶液系統下,以塗佈術接管的方式降低電滲流使硼酸被滯留於液體接合槽中;在毛細膠體電泳系統中,添加的PEO分子則藉由液體接合槽的使用維持分離效率亦可避免其進入質譜。使用雙槽式介面後成功銜接前述兩個毛細電泳系統,並利用質譜辨別鍵結異構物的結構。 Separation of glycans is necessary before analyzed by MS for their structural complexity. A comprehensive study of glycan structure could be improved by analyzed though CE-MS. The glycans of RNase B, categorized as high-mannose type oligosaccharides, were chosen as the model compounds. The glycans released from RNase B were labeled with p-aminobenzoic acid (ABA) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS) either by reductive or non-reductive amination. Two kinds of electrophoresis system, including CZE using borate buffer and CGE using acetate buffer with polyethylene oxide (PEO), were selected to analyze the glycans. In borate buffer system, normal polarity was employed under high pH environment. In the acetate buffer with polyethylene oxide (PEO), the reverse polarity was employed under low pH environment. According to the premier results using CE-UV, in both CZE and CGE system, the better separation can be obtained for glycans labeled with APTS by reductive amination can be obtained with better separation results comparing to glycans labeled with APTS by non-reductive amination and ABA labeling either by reductive or non-reductive amination. To avoid ion suppression by borate ions and PEO as CE was coupled to MS, a double junction interface was applied. By employing the double junction interface, which was composed of a liquid junction, a connecting column and a low-flow interface, borate and PEO were restricted in the liquid junction, while the analytes were detected successfully and the isomers structure of M7G2 were analyzed by MS. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27829 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
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