Gceims

Linkage analysis

Carbohydrate sequence and anomeric configurations determination

Strategy dedicated to the determination of the structures of the W-glycans of plant glycoproteins. This strategy can be applied to a purified glycoprotein or a mixture of glycoproteins. Reduced and carboxamidomethylated glycoproteins are submitted to a proteolytic digestion to generate small peptides and gly-copeptides before PNGase A digestion. PNGase A released glycans are separated from peptides using a Sep-Pak C18 cartridge and desalted on nonporous graphi-tized carbon solid-phase extraction cartridge, PNGase A-released W-glycans are then characterized by MALDI-MS before and after on-plate sequential exoglycosidase digestions. Their methylated derivatives are also characterized by MALDI-MS before and after sequential exoglycosidase digestions and by linkage analysis. Structural assignments are based on molecular weight, susceptibility to exoglycosidase digestions, and linkage data.

monosaccharides were attached. Structural assignments of PNGase A-released glycans are based on MW, susceptibility to exoglycosidase digestions, and linkage data.

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