Normal phase glycan analysis by HPLC resolves glycans based on hydrophilicity, which is determined primarily by size, as well as arm specificity and linkage. It is one of the most widely used methods used for glycoprofiling a mixture of 2-AA and 2-AB labeled glycans.
N-linked glycans are released from glycoproteins via enzymatic cleavage, most often by the addition of PNGase F. Alternatively, our Endo F Multi-kit can be used to deglycosylate native proteins that are resistant to PNGase F cleavage under non-denatured conditions. The Endo F enzymes are known to be less sensitive to glycan location within the protein’s three-dimensional structure. It should be noted that these three enzymes cleave N-linked oligosaccharides between the two GlcNAc residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. The remaining monosaccharide imparts a charge, thereby increasing the solubility of the protein. In contrast, PNGase F removes the oligosaccharide intact.
For O-linked glycan release, the Ludger Liberate Orela Kit allows the release of glycans leading a free reducing terminii suitable for fluorescent labelling. The kit is straightforward and contains a simple method with no special handling techniques as required by the alternative method, hydrazinolysis.
The released N-glycans can then be purified using EC50 cartridges while CEX cartridges can be used for the purification of O-linked glycans. These cartridges bind the glycans, while proteins, salts and detergents that can interfere with subsequent labeling with 2-AB or 2-AA fluorescent molecules are removed. After fluorescent labeling excess fluorescent label can be removed with S cartridges, or T1 cartridges for high throughput applications.
Subsequent HILIC or HPLC analysis is performed using LudgerSep normal phase columns and buffers.