Glycan Sequencing

To fully understand the biological relevance of a particular glycan in a glycoconjugate one needs to elucidate the primary sequence and subsequent structure. Minor changes in carbohydrate structure can greatly influence the bioactivity of a particular glycoprotein. The high diversity of glycosylation makes structural elucidation very difficult and in most cases a combination of orthogonal methods is used for an accurate glycan profile.  Frequently used analytical methods for the structural analysis of complex carbohydrates includes capillary electrophoresis (CE), mass spectrometry (MS) and high-performance liquid chromatography (HPLC), often combined with exoglycosidase digestion techniques.

• Sequencing glycans typically begins with the release of the N-linked carbohydrate moieties from the glycoprotein using an endoglycosidase, such as PNGase F.
• 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.
• Modified glycans can be analyzed by mass spectrometry, and putative structures can be assigned based on the mass spectral data.
• Exoglycosidases can be used to elucidate ambiguous structures in a glycan profile. Most exoglycosidases are specific for the sugar and the anomericity (α or β) of the linkage that they will recognize and cut. The researcher can use specific exoglycosidases to determine the anomericity and the type of terminal sugar residues present on the glycan being analyzed.