Combination of beta-elimination and liquid chromatography/quadrupole time-of-flight mass spectrometry for the determination of O-glycosylation sites.

Determination of O-glycosylation sites in glycopeptides was developed by using two model compounds designed from mucin2 tandem repeat motif and erythropoietin. beta-Elimination/addition reaction using dimethylamine on glycosylated site through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. The use of dimethylamine was efficient to release the O-linked glycan in a reaction time period of 2-6h at 55 degrees C. Peptide sequencing was then performed using the liquid chromatography/quadrupole time-of-flight mass spectrometry and MS-MS experiments. Interpretation of fragmentation pathways of the beta-elimination/addition products enabled straightforward recognition of glycosylation site. Compared to the fragmentation of corresponding native peptides, mass shift of -18 Da or +27 Da was clearly observed for the two kinds of beta-elimination/addition products of the glycosylated threonine. Dimethylamine was found to provide higher efficiency of beta-elimination/addition than methylamine and ammonia.