Roger A. O'Neill

Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis

For most separations-based analyses of glycoprotein oligosaccharides, the first step is release of the oligosaccharides from the polypeptide. Historically, O-linked and N-linked oligosaccharides have been released from glycoproteins using chemical means, such as alkaline degradation (beta-elimination) or hydrazinolysis. In the last two decades, a growing repertoire of enzymes, including endoglycosidases and glycoamidases, able to release glycoprotein oligosaccharides under mild conditions, have become available. This review traces the discovery, characterization and use of these glycoprotein oligosaccharide releasing enzymes. Emphasis is placed on providing information of practical value for the researcher wishing to incorporate enzymatic oligosaccharide release into their study of glycoprotein oligosaccharide structure and function.

Multi-dimensional mapping of pyridylamine-labeled N-linked oligosaccharides by capillary electrophoresis

A simple, sensitive and reproducible multi-dimensional capillary electrophoresis (CE) oligosaccharide mapping method is reported. The structures of 20 identified N-linked oligosaccharides have been assigned mapping positions from which co-migrating unknown oligosaccharides can be characterized. The separation protocols developed have been demonstrated to separate both charged and neutral oligosaccharides. One dimension involves electroendosmotic flow-assisted CE in a sodium acetate buffer, pH 4.0. A second dimension involves separation based on borate complexation electrophoresis in a polyethylene glycol-containing buffer. A third dimension developed specifically for neutral oligosaccharides, using a sodium phosphate buffer, pH 2.5, has been shown to resolve neutral species not able to be separated by the other two dimensions. Thus, a three-dimensional map was generated to facilitate structural characterization of these oligosaccharides.

A Facile Method for the Release, Labeling and ce Analysis of Glycoprotein Oligosaccharides

Publisher Summary This chapter describes a facile method for the release, labeling, and capillary electrophoresis (CE) analysis of glycoprotein oligosaccharides. It describes a finger printing method in which N-linked oligosaccharides of glycoproteins are rapidly and effectively released and labeled, facilitating their analytical separation by high-performance liquid chromatography (HPLC) or CE. In this method, oligosaccharides are enzymatically released using peptide- N -glycosidase F, followed by chemical derivatization with the chromophore l-phenyl-3-methyl-5-pyrazolone. PNGase F releases accessible Asn-linked oligosaccharides by cleaving β-aspartylglucosylamine bonds, resulting in the release of intact oligosaccharides. During the process, the asparagine residue to which the carbohydrate was attached is converted to aspartic acid. The protocol developed requires no purification of the released oligosaccharides prior to labeling, and the oligosaccharides once labeled are ready for analysis by CE and/or HPLC immediately following a simple liquid–liquid extraction used for their clean-up. Increasing the PNGase F concentration up to 10-fold did not increase the extent of deglycosylation past the end point that was rapidly achieved with much less enzyme.