- 學位論文
高甘露醣鏈於非揮發性環糊精和硼酸緩衝溶液之毛細電泳質譜研究
CE-MS Analysis of High-Mannose Glycans by Using Nonvolatile Sulfated β-Cyclodextrin and Borate Buffer
摘要
核糖核酸酶B醣鏈結構含有M5G2、M6G2、M7G2、M8G2、M9G2(M,Man; G,GlcNAc)五種，其中M7G2具有三種結構異構物。進行醣鏈與醣鏈異構物之結構分析時，需要先利用層析方法將其分離。實驗中，分別以對胺基苯甲酸乙酯(ABEE)和對胺基苯甲酸(ABA)標記高甘露醣鏈，嘗試利用毛細管區帶電泳系統於非揮發性硼酸鹽緩衝溶液分離不同大小的醣鏈與醣鏈異構物。於硼酸鹽緩衝溶液中，對胺基苯甲酸(ABA)標記高甘露醣鏈具有較佳分離效果，但仍無法達到高甘露醣鏈達基線分離與M7G2三種同分異構物之分離。利用添加負電荷環糊精於硼酸緩衝溶液系統，雖然M7G2三種同分異構物仍未達到分離，不同大小的高甘露醣鏈可達基線分離。環糊精和硼酸鹽緩衝溶液對於負離子電噴灑會有嚴重訊號抑制以及電噴灑時會因結晶而造成噴頭阻塞與噴灑不穩定。因此本研究使用實驗室所開發之低鞘流-液體接合式介面以克服上述問題。此介面包含三大部分：液體接合槽、銜接管及低鞘流介面。實驗中將銜接管以塗佈的方式降低電滲流，使得硼酸根離子和環糊精會停留於液體接合槽中，以避免硼酸離子與環糊精進入質譜造成分析物訊號抑制，但仍保有分離效果。
並列摘要
Ribonulease B contains five high-mannose oligosaccharide which are M5G2, M6G2, M7G2, M8G2 and M9G2 (M, Man; G, GlcNAc). Among these glycan, the M7G2 consists of three structural isomers. The separation of these glycan isomers is necessary to characterize its structure. CE was chosen as separation platform because of its high separation efficiency. In our experiment, high-mannose glycans were labeled with p-aminobenzoic acid ethyl ester (ABEE) and aminobenzoic acid (ABA) resepectively, then these derivatives were analyzed by CE under nonvolatile borate buffer system. Our results indicated that separation efficiency was better for ABA-labeled glycans. The high-mannose glycans were partially separated, whereas three isomers of M7G2 coeluted under this condition. To achieve better separation efficiency, sulfated-β-cyclodextrin was added into the borate buffer system. The results show that the high-mannose glycans were baseline separated. However, these nonvolatile species can suppress the analyte signals and crystallize to block the sprayer to terminate ESI process. A Liquid-Junction/Low-Flow interface was utilized to overcome the above problems. The interface consists of a liquid junction reversoir, a coated connecting column and a low flow interface. A coated connecting column was employed to reduce EOF. The coating solutions contained 10% polybrene solution can reduce the EOF (18%) in comparison with non-modified capillary. Under such condition, the borate and cyclodextrin can retain in the liquid-junction revervoir, whereas the analyte can migrate toward the ESI source due to EOF. With this approach, the signals of analyte were not suppressed and the separation efficiency was maintained.