Determining the Structure of Oligosaccharides N‐ and O‐Linked to Glycoproteins

Abstract

Many proteins involved in biological events are glycosylated. A glycoprotein consists of a mixture of glycosylation variants of a single polypeptide chain, known as glycoforms. It has become clear that a detailed understanding of the roles which glycosylation plays in the biosynthesis, transport, biological function, and degradation of a glycoprotein can only be achieved when the protein and sugar(s) are viewed as an entity. Many glycoproteins can now be modeled by combining glycan sequencing data and oligosaccharide structural information with protein structural data. Pivotal to this approach is sensitive, state-of-the-art oligosaccharide sequencing technology which can give a rapid insight into the glycosylation of a glycoprotein without the need for sophisticated equipment and expertise. This unit gives a detailed introduction into the analysis of glycans, and the many figures will help the user identify which type of experiment needs to be undertaken. Methods for releasing glycans from glycoproteins are followed by protocols for labeling and purifying (by HPLC) the glycans from the rest of the components. Strategies for N- and O-glycan analysis are also included.

Glycosylation analysis of Chinese hamster ovary produced glycoproteins

“…The glycan elu tion peaks are compared with the retention time of a dextran ladder (increasing linear glucose units) using a fifth order polynomial standard curve of glucose unit (GU) values versus retention time ( Figure 3). Prelimi nary identification of the structures uses a comparison of the GU values of the glycans to a standard data base of GU of glycans (such as Glycobase) [61,63,[65][66]. Further analysis using digestion arrays of glycans with exo glycosidases can confirm structure and linkages by the sequential removal of specific sugar residues mon itored by shifts in retention times or GU values [61].…”

N-glycan profiling of bovine follicular fluid at key dominant follicle developmental stages

“…The gradient conditions were as follows: a linear gradient of 0-5% solvent A over 12 min at a flow rate of 1 ml/min, followed by 5-21% solvent A over 13 min and then 21-50% solvent A over 25 min, 80-100% solvent A over 5 min and then 5 min at 100% solvent A. The samples were injected in water, and a fetuin N-glycan standard was used for calibration (Royle et al 2006).…”

“…To determine the sequence, monosaccharide type and linkage of sugar residues as described previously (Royle et al 2006), whole pools and AEC-fractionated 2-AB-labelled N-glycans were digested with the combinations of linkage-specific exoglycosidase enzymes before separation on HPLC. The protocol used the following enzymes (with the specificities and activities in brackets) obtained from Prozyme, San Leandro, CA, USA: NAN1 sialidase (EC 3.2.1.18, releases a2-3 sialic acid, 1 U/ml); ABS, Arthrobacter ureafaciens sialidase (EC 3.2.1.18, releases a2-3,6,8, 9 sialic acid, 1 U/ml);…”

High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

“…The relative amount of the peaks is consistent with that reported for IgG1 glycan structures. 42 Because the effect of the two isomeric structures is unclear, we report the sum of G1 (H4N4F) and G1-F (H4N4), respectively.…”