Manzi A E
University of California San Diego, La Jolla, California, USA.
Curr Protoc Mol Biol. 2001 May;Chapter 17:Unit17.19A. doi: 10.1002/0471142727.mb1719as32.
Once the presence of carbohydrate in a glycoprotein has been confirmed, the next step is to determine the precise molar ratio of its monosaccharide constituents. The analysis involves two major phases. The release of the individual monosaccharides is achieved by methanolysis (described here), total acid hydrolysis, or enzymatic release of sialic acids. The resulting mixtures of monosaccharides are then analyzed by methods described in this unit: fractionation, characterization, and quantitation by high-performance liquid chromatography using anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and other HPLC systems or by gas-liquid chromatography with flame ionization detection. The identity of the individual monosaccharides is determined by comparison with known standards processed and analyzed in the same way.
一旦确定糖蛋白中存在碳水化合物，下一步就是确定其单糖成分的精确摩尔比。分析包括两个主要阶段。通过甲醇解（此处所述）、完全酸水解或唾液酸的酶促释放来实现单个单糖的释放。然后，使用带有脉冲安培检测的阴离子交换色谱法的高效液相色谱法（HPAEC-PAD）和其他HPLC系统，或通过带有火焰离子化检测的气液色谱法，按照本单元所述方法对所得单糖混合物进行分析：分级分离、表征和定量。通过与以相同方式处理和分析的已知标准品进行比较，确定各个单糖的身份。
