Chen F T, Evangelista R A
Beckman Coulter Inc., Fullerton, CA 92835, USA.
Electrophoresis. 1998 Nov;19(15):2639-44. doi: 10.1002/elps.1150191512.
A method for analysis of N-linked oligosaccharides derived from glycoproteins including sialic acid-containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. Glycoproteins were heat-denatured in the presence of a reducing agent and the N-linked oligosaccharides were released by peptide N-glycosidase (PNGase F; EC3.5.1.52)-catalyzed hydrolysis. The released N-linked oligosaccharides were derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N-linked oligosaccharide, desialylated, galactosylated biantennary, core-substituted with fucose (A2F) was tested for APTS-based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N-linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resolution of the N-linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.
本文介绍了一种分析糖蛋白中N-连接寡糖（包括含唾液酸的种类）的方法。该方法基于特定化学和酶促转化与毛细管电泳（CE）分离及激光诱导荧光（LIF）检测相结合。糖蛋白在还原剂存在下进行热变性，然后通过肽N-糖苷酶（PNGase F；EC3.5.1.52）催化水解释放N-连接寡糖。在温和的还原胺化条件下，用8-氨基芘-1,3,6-三磺酸盐（APTS）对释放的N-连接寡糖进行衍生化，使唾液酸去除和岩藻糖残基损失最小化。对一种模型N-连接寡糖（去唾液酸化、半乳糖基化的双天线型，岩藻糖核心取代，即A2F）进行了基于APTS的衍生化化学测试，加合物回收率良好，且未损失岩藻糖和神经氨酸残基。报道了从胎球蛋白、重组人促红细胞生成素和激肽释放酶衍生的高度唾液酸化N-连接寡糖的图谱，数据表明本方法能为糖蛋白衍生聚糖的指纹识别提供高分辨率的N-连接寡糖图谱。
