N-glycosylation of ovomucin from hen egg white

Ovomucin is a bioactive egg white glycoprotein responsible for the gel properties of fresh egg white and is believed to be involved in egg white thinning, a natural process that occurs during storage. Ovomucin is composed of two subunits: a carbohydrate-rich β-ovomucin with molecular weight of 400–610 KDa and a carbohydrate-poor α-ovomucin with molecular mass of 254 KDa. In addition to limited information on O-linked glycans of ovomucin, there is no study on either the N-glycan structures or the N-glycosylation sites. The purpose of the present study was to characterize the N-glycosylation of ovomucin from fresh eggs using nano LC ESI-MS, MS/MS and MALDI MS. Our results showed the presence of N-linked glycans on both glycoproteins. We found 18 potential N-glycosylation sites in α-ovomucin. 15 sites were glycosylated, one site was found in both glycosylated and non-glycosylated forms and two potential glycosylation sites were found unoccupied. The N-glycans of α-ovomucin found on the glycosylation sites are complex-type structures with bisecting N-acetylglucosamine. MALDI MS of the N-glycans released from α-ovomucin by PNGase F revealed that the most abundant glycan structure is a bisected type of composition GlcNAc6Man3. Two N-glycosylated sites were found in β-ovomucin.