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.