Glycan enzymatic release

Structural analysis of A/-linked oligosaccharides is typically conducted by their enzymatic release from the protein, derivatization with a suitable fluorescent tag, and CE separation. A variety of oligosaccharide standards have been developed to facilitate identification of unknown species based on migration time comparison [60,138], For further structural characterization, glycans can be fractionated and subjected to LC-ESI-MS or MALDI-TOF-MS analysis [139-141], Alternatively, online CE-MS analysis is feasible as described in Section 7.3.1. 

Glycans can be removed by either chemical or enzymatic methods. The advantages of enzymatic release are that (a) no chemical alterations are made to the sugars and (b) the glycans retain a free reducing terminal that can easily be labeled with a fluorophore for highly sensitive detection. 

For glycan analysis, the glycans are released by chemical or enzymatic methods. The prevailing analytical methods for glycan profiling involve some form of labeling... 

Fig. 3. Chemical and enzymatic treatment to reduce the size and complexity of the GPI anchor for mass spectrometric analysis. PIPLC removed the acylalkylglycerol lipids, then endoproteinase Lys C cut the amino acid chain after Lys220, giving the C-terminal peptide attached to the phosphorylated glycan. Incubation with 50% aqueous HF was used to hydrolyze the phosphodiester bonds and to release the glycan and the peptide, which were separated by RP-HPLC and analyzed independently.

Immobilization approach. The glycan sample is immobihzed to a solid support. The immobilized glycan is then exposed to exoglycosidases sequentially and the monosaccharides released by each enzymatic digestion is detected, identified and quantified. 

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