Highly Sensitive Methods Using Liquid Chromatography and Capillary Electrophoresis for Quantitative Analysis of Glycoprotein Glycans

摘要

This review summarizes our recent works concerned on the analysis of glycoprotein glycans. Glycoprotein glycans are highly complicated and have variations that number into the thousands. Therefore the method for their analyses should be carefully chosen according to the complexity and quantity of glycans in samples. To shorten the time for the preparation of labeled glycans from glycoprotein, we proposed two methods: PNGase F/NBD-F method enables the preparation of fluorescently labeled glycans from glycoproteins about 2 hr. A combination of a neutrally coated capillary and borate buffer eliminates the purification steps for the removal of excess reagents from the glycan samples for capillary electrophoresis. Partial filling affinity capillary electrophoresis using glycan-recognizing proteins such as lectins, glycosidases and immunoglobulins, was used for the structural analysis of glycans. The method was applied to the specific detection of NeuGc and α-Gal containing glycans known as the heterogenic antigens. Sensitivity of the detection of glycoprotein glycans separated by microchip-based electrophoresis was enhanced by in situ fabricated sufonate-type polyacrylamide gel. This method enhances the sensitivity by a factor of 10~(5). In situ fabrication of lectin impregnated gels enables specific entrapment and analysis of glycans. Conversion reaction from fluorescently labeled glycans (1-amino-1-alditols) to free oligosaccharides will be helpful for the functional analysis of glycans.