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.
Zheng
Yufang
Y
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong SAR, PR China.
Guo
Zhihong
Z
Cai
Zongwei
Z
D001665:Binding Sites; D002853:Chromatography, Liquid; D004921:Erythropoietin; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods; D055262:Mucin-2; D013912:Threonine
D006020:Glycopeptides; D055262:Mucin-2; D004921:Erythropoietin; D013912:Threonine