Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis
Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis
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DOI：
10.1016/j.jchromb.2011.08.026
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发表时间：
2011-10-01
影响因子：
3
通讯作者：
Suzuki, Shigeo
中科院分区：
文献类型：
--
作者：
Oyama, Takehiro;Yodohsi, Masahiro;Suzuki, Shigeo
在肽 N(4)-(N-乙酰基-β-D-葡萄糖胺基)天冬酰胺酰胺酶 F (PNGase F) 作用的第一步中，天冬酰胺型寡糖作为 N-糖胺从核心蛋白中释放出来。因此，释放的 N-糖胺型寡糖（至少在酶反应过程中专门存在）可以用胺标记试剂衍生化。在这里，我们报告了一种使用 4-氟-7-硝基-2,1,3-苯并恶二唑 (NBD-F) 作为糖胺型寡糖标记试剂的方法。我们应用该方法对一些源自已充分表征的糖蛋白的寡糖混合物进行了灵敏分析，包括人转铁蛋白、α(1)-酸性糖蛋白、牛胎球蛋白和核糖核酸酶 B。NBD 标记的寡糖在酰胺键合柱或二醇硅胶柱上成功分离。该标记方法包括糖蛋白中寡糖的释放和寡糖的衍生化，一锅反应，在3小时内完成。该方法的灵敏度比在 HPLC 中使用荧光检测的对氨基苯甲酸乙酯 (ABEE) 标记的方法高出大约五倍，并且在 ESI-LC/MS 中的响应高出一个数量级。我们还应用这种方法通过毛细管电泳和激光诱导荧光检测 (LIF-CE) 对糖蛋白衍生的寡糖进行灵敏分析。 HPLC 和 LIF-CE 的检测限分别为 100 fmol 和 4 fmol。 (C) 2011 Elsevier B.V. 保留所有权利。
Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N(4)-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, alpha(1)-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amidebonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3 h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100 fmol and 4 fmol, respectively. (C) 2011 Elsevier B.V. All rights reserved.