Hayase T, Sheykhanazari M, Bhavanandan V P, Savage A V, Lee Y C
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Anal Biochem. 1993 May 15;211(1):72-80. doi: 10.1006/abio.1993.1235.
A high-pH anion-exchange chromatography (HPAEC) system with a pulsed amperometric detector was developed for analysis of O-linked oligosaccharides in glycoproteins. Since O-linked oligosaccharides are usually released from glycoproteins as alditols by beta-elimination with reduction and they are retained less on an anion-exchange resin than their reducing counterparts, a special set of HPAEC conditions optimized for separation of sialyl O-linked oligosaccharides different from those for N-linked oligosaccharide separation had to be devised. HPAEC separation of desialylated O-linked oligosaccharides is even more difficult because of the lack of carboxylic acids or anomeric hydroxyls. Therefore, a precolumn derivatization technique was developed for these oligosaccharides. Reduced desialylated oligosaccharides were de-N-acetylated followed by N-succinylation, which endowed a negative charge at each amino sugar residue in the oligosaccharides. The desialylated oligosaccharides are derivatized in the same vial and can be easily separated on HPAEC. The new HPAEC technique presented here promises to be a valuable tool for analysis of O-linked oligosaccharides in glycoproteins.
开发了一种配备脉冲安培检测器的高pH值阴离子交换色谱（HPAEC）系统，用于分析糖蛋白中的O-连接寡糖。由于O-连接寡糖通常通过还原β-消除反应从糖蛋白中以糖醇形式释放出来，并且它们在阴离子交换树脂上的保留能力比其还原对应物弱，因此必须设计一套专门针对唾液酸化O-连接寡糖分离优化的HPAEC条件，这与N-连接寡糖分离的条件不同。由于缺乏羧酸或异头羟基，去唾液酸化O-连接寡糖的HPAEC分离更加困难。因此，针对这些寡糖开发了一种柱前衍生技术。还原的去唾液酸化寡糖先进行脱N-乙酰化，然后进行N-琥珀酰化，这使得寡糖中的每个氨基糖残基都带有负电荷。去唾液酸化寡糖在同一小瓶中进行衍生化，并且可以在HPAEC上轻松分离。本文介绍的新HPAEC技术有望成为分析糖蛋白中O-连接寡糖的有价值工具。
