Rapid de-O-glycosylation concomitant with peptide labeling using microwave radiation and an alkyl amine base.
Procedures are detailed for a quantitative release of O-linked glycans from peptides that now provide a shorter reaction time, a possible identification of O-linked sites, and a quantification of all reaction products. The release was initiated by a mild base, dimethylamine, and accelerated by microwave radiation. Differential analysis using standard glycoproteins has shown improved release efficiency concurrent with facile incorporation of dimethylamine into the former O-linked sites. In situ glycan reduction insures protection against peeling and is synchronous with subsequent studies by high performance MS(n) sequencing. The protocols were established with a synthetic O-GlcNAc peptide that would mimic the linkage chemistry and applied to a well characterized glycoprotein bovine fetuin with both N- and O-linked glycans and a highly glycosylated swine mucin.
Maniatis
Stephanie
S
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, USA.
Zhou
Hui
H
Reinhold
Vernon
V
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004123:Dimethylamines / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D008872:Microwaves; D010455:Peptides / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D013552:Swine
D004123:Dimethylamines; D006023:Glycoproteins; D010455:Peptides; D011134:Polysaccharides; C034516:dimethylamine