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

Abstract

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.

Accurate Quantification of N-Glycolylneuraminic Acid in Therapeutic Proteins Using Supramolecular Mass Spectrometry

“…Overall, there was good agreement between the quantitation of the sialic acids in glycoproteins and the values reported in the literature, which indicates excellent accuracy and precision of the analytical method (the details are available in the Supporting Information, Figure , and Table S6). ,− …”

Development of Microfluidic Components for Micro Total Analysis Systems

“…The microfluidic scale has several advantages, such as lower consumption of reagents, separation speed and resolution, device portability, high repeatability, multiple assay etc. For this reason, many applications of microfluidic systems have been proposed so far, not only in chemical analysis but also in medical diagnosis, and the food and agricultural industries [4][5][6][7][8].…”

Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

“…Saccharides are one of the most abundant biological molecules that are widely distributed in nature. Most saccharides are neutral and highly hydrophilic, and do not possess chromophoric or fluorimetric properties [1][2][3]. Therefore, numerous fluorescent amines have been developed to derivatize saccharides by reductive amination and improve their sensitivity and resolution in both HPLC and capillary electrophoresis (CE) analyses [4][5][6][7][8].…”