Chen F T, Dobashi T S, Evangelista R A
Beckman Instruments, Inc., 4300 Harbor Boulevard D-14E, Fullerton, CA 92834, USA.
Glycobiology. 1998 Nov;8(11):1045-52. doi: 10.1093/glycob/8.11.1045.
A method for quantitative analysis of monosaccharides including N-acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N-acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N-acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS-monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.
本文提出了一种对包括源自含唾液酸寡糖和糖蛋白的N-乙酰神经氨酸在内的单糖进行定量分析的方法。该分析基于化学和酶法相结合，并与毛细管电泳（CE）分离和激光诱导荧光（LIF）检测联用。本方法采用了一种简化的酸水解程序，包括温和水解（0.1 M三氟乙酸）以释放唾液酸，以及强酸水解（2.0 N三氟乙酸）以生成氨基糖和中性糖。寡糖和糖蛋白经强酸水解释放出的氨基糖在氰基硼氢化钠存在下与中性糖一起进行重新乙酰化，并与8-氨基芘-1,3,6-三磺酸盐（APTS）衍生化，以定量生成高度稳定的荧光APTS加合物。N-乙酰神经氨酸（Neu5Ac）是大多数哺乳动物糖蛋白的主要成分，通过神经氨酸醛缩酶（N-酰基神经氨酸丙酮酸裂解酶，EC 4.1.3.3）的作用在快速特异性反应中转化为N-乙酰甘露糖胺（ManNAc）和丙酮酸。然后，ManNAc以与其他单糖相同的方式用APTS进行衍生化。该方法已用于定量纯Neu5Ac以及6'-唾液酸基-N-乙酰乳糖胺和牛胎球蛋白聚糖经温和酸水解得到的产物。在七种其他单糖的混合物中，从已知量的Neu5Ac或从糖蛋白中存在的唾液酸化寡糖中均可定量回收N-乙酰甘露糖胺。程序顺序包括酸水解、酶转化和APTS衍生化，可定量回收APTS-单糖加合物。经APTS衍生化并通过CE-LIF检测的糖的检测限，Neu5Ac为100 pmol，其他糖为50 pmol。
