To fully understand the biological relevance of a particular glycan in a glycoconjugate one needs to elucidate the primary sequence and subsequent structure. Although the expression levels of the enzymes needed to synthesize and process glycans (glycosidases and glycosyltransferases) can be measured, the synthetic pathways are not linear and a single compound can be used as a substrate in multiple reactions, making it difficult to accurately determine the reaction’s outcome. Therefore, it becomes impossible to predict the precise sequence and structures of glycans produced by a particular cell.
The first analytical step is typically deglycosylation. Free glycans can be analyzed directly, but they are often derivatized with fluorescent markers such as 2-aminobenzamine (2AB) or with methyl groups (permethylation) to increase the sensitivity of detection, which is essential to observe low abundance glycan species. These modified glycans can be analyzed by mass spectrometry and putative structures can be assigned based on the mass spectral data.
Learn about glycobiology and its importance in clinical and diagnostic applications in this episode of NEB TV. Also, hear more about how NEB is setting the bar for product quality in this rapidly growing field.
Learn about the core sequences and common modifications of N-linked and O-linked glycans in this video. Analysis of these glycans can be accomplished with the use of deglycosylation enzymes, which can provide complete sugar removal with no protein degradation.
Learn how glycosidases are used to analyze multiple glycoprotein samples. Here, the model glycoprotein, hCGβ, which carries both N- and O-glycans, is demonstrated.
Learn more about the structural elucidation of complex and diverse heparin oligosaccharides using Bacteroides Heparinase I, II and III in combination with downstream mass spec analysis.