Characterisation of individual N-and O-linked glycosylation sites using Edman degradation

摘要: Publisher Summary Solid-phase Edman degradation provides the ideal chemical method for purification of individual glycoforms as it is not limited by problems associated with endoglycosidase specificity. This chapter describes techniques determining sites N and O-glycosylation a part in automated solid phase micro-sequencing glycoproteins. Sites O- N-linked glycosylation can be positively identified during solid-phase if polar solvents, such anhydrous trifluoroacetic acid (TFA), are used to extract cleaved amino from reaction cartridge. The oligosaccharide released labeled reducing terminal sugar, phenylthiohydantoin (PTH)-glycoamino acid. Although, there several excellent methods analysis N- O-linked sites, particularly, LC-ionspray MS analysis; however, glycopeptides that contain domain clustered choice precise site identification characterization. Thus, combination carbohydrate analysis, high performance anion exchange chromatography (HPAEC) ionspray mass spectrometry, will allow new approach characterization heavily glycosylated proteins previously thought intractable protein chemistry studies.