Glycosidic bonds rarely affect the function of glycoproteins, so they usually do not need to be determined. However, the glycan structures of glycoproteins are highly complex, and assessing the configuration and position of N-glycosidic bonds can be helpful for analyzing the structure of glycoproteins. Five common types of N-glycan bonds are known, with the most common being the linkage of N-acetylglucosamine to asparagine (GlcNAcβ1-Asn). Other types linked to Asn include glucose in the laminin of mammals and archaea, N-acetylgalactosamine (GalNAc) in archaea, and rhamnose-containing bacteria. Reports also indicate that glucose links to arginine in sweet corn.
HILIC-UHPLC Analysis of N-glycan Bonds
N-glycans are hydrolyzed from glycoproteins using Peptide N Glycosidase F (PNGase F). The hydrolyzed N-glycans are labeled with 2-aminobenzamide (2-AB) and then digested using various exoglycosidases. Finally, glycans are analyzed using hydrophilic interaction chromatography (HILIC) combined with fluorescence and mass spectrometry detection.
General Steps for N-glycan Bond Analysis
1. Sample Preparation
2. N-glycan Hydrolysis
3. 2-AB Labeling and Sequential Reaction of Labeled Glycans with a Series of Exoglycosidases
4. Analysis Based on UPLC-HILIC-FLD
5. Additionally, detailed glycan structures can be determined via MS/MS fragmentation
Role of Endoglycosidases:
Endoglycosidases can hydrolyze oligosaccharides from glycoproteins or glycolipids. Typically, they do not hydrolyze oligosaccharides from bound protein and lipid molecules. Endoglycosidases function by cleaving the glycosidic bond between two sugar monomers in the polymer.
Endoglycosidase H: Hydrolyzes oligomannose and hybrid N-glycans
Endoglycosidase F: Hydrolyzes simple biantennary N-glycans
