Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method
Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method
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DOI：
10.1016/j.chroma.2005.08.052
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发表时间：
2006-02-17
影响因子：
4.1
通讯作者：
Kakehi, K
中科院分区：
文献类型：
--
作者：
Kamoda, S;Nakanishi, Y;Kakehi, K
毛细管电泳（CE）是一种高分辨率分析糖蛋白衍生的碳水化合物混合物的有效工具。然而，CE具有将几纳升的样品溶液注入到狭窄毛细管中的缺点。因此，我们必须制备高浓度的样品溶液用于CE分析。本研究采用头柱场放大样品堆积法对糖蛋白中N-连接寡糖进行了双向电泳分析。模型研究表明，我们实现了。60-360倍浓度对3-氨基苯甲酸（3-AA）标记糖链的分析有影响。将该方法应用于糖蛋白中N-连接寡糖的分析。通过2D-PAGE检查油酸性糖蛋白（AGP）的杂合，即糖型，并通过PNGase F的凝胶内消化释放N-连接寡糖。释放的寡糖用3-AA衍生化并通过CE分析。结果表明，具有较低pI值的糖型含有大量的四和三前体寡糖。相比之下，具有较高pI值的糖型含有丰富的双触角寡糖。结果清楚地表明，通过考马斯亮蓝染色在2D-PAGE凝胶上检测到的糖蛋白糖型斑点足以用于寡糖的定量分析。(c)2005 Elsevier B. V.保留所有权利。
Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved. 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of oil-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antermary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform, detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides. (c) 2005 Elsevier B.V. All rights reserved.