Abstract
BackgroundIn sharp contrast with analysis of N-glycan that can be prepared by PNGase F, O-glycan analysis remains challenging due to a lack of versatile and simple procedures, especially those mediating cleavage of O-glycans from proteins. Most N-glycans and O-glycans are modified with sialic acids at the non-reducing end and their glycosidic linkages are labile, making it difficult to measure glycans by mass spectrometric analysis. In addition, sialic acid residues present on glycan chains via α2,3-, α2,6-, and α2,8-linkages as structural isomers. ResultsIn this study, we firstly established a direct and linkage-specific derivatization method for sialylated O-glycans on proteins via linkage-specific lactone-opening aminolysis. In this procedure, labile sialylated glycans were not only stabilized, but also allowed distinguishing between sialyl linkages. Furthermore, we revealed that general reductive β-elimination was not useful for O-glycan cleavages with undesirable degradations of resulting methyl amides. Using β-elimination in the presence of pyrazolone (PMP), with low pH despite alkali base concentration, SALSA-derivatized O-glycans could be cleaved with minimal degradations. Cleaved and PMP-labeled O-glycans could be efficiently prepared in an open reaction system at high temperature (evaporative BEP reaction) and detected by simple liquid-phase extraction. Moreover, in the evaporative BEP reaction by changing the alkali solution with LiOH, the lithiated O-glycans could be observed and provided a lot of fragment information reflecting the complex structure of the O-glycans. SignificanceDirect sialic acid linkage-specific derivatization of O-glycans on glycoproteins is simple protocol containing in-solution aminolysis-SALSA and acetonitrile precipitation for removal of excess reagents. Evaporative β-elimination with pyrazolone makes possible intact O-linked glycan analysis just by liquid-phase extraction. These analytical methods established by the appropriate combination of direct-SALSA and evaporative β-elimination will facilitate O-glycomic studies in various biological samples.
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