Site‐specific N‐linked glycosylation analysis on the human salivary mucin MUC5B using Precursor Ion Discovery on the CAPLC Q‐TOF system

Abstract

Introduction Mucins are large highly O‐glycosylated oligomeric glycoproteins that form the polymer matrix of mucus, which forms part of the innate immune system. MUC5B, the predominant gel‐forming mucin in saliva, is around 600 kDa in mass before glycosylation. O‐linked glycosylation is abundant in defined repeated domains and may account for 80% of the observed mass of the secreted. Not much is known about the N‐linked glycosylation, although there are at least 34 potential sites. Despite little being known about the structure, N‐glycosylation has been shown to be important but not essential for mucin synthesis (Perez‐Vilar & Hill 1999). Here, we aim to study the N‐linked glycosylation site specifically using mass spectrometryMaterials and methods A sample of purified human MUC5B mucin was digested with trypsin and separated by size exclusion chromatography (Thornton et al. 1999). The previously uncharacterized fractions were analysed by online LC Q‐Tof MS in order to characterize any N‐glycosylated peptides present. Glycopeptide discovery was steered by the Precursor Ion Discovery function, which detects potential glycopeptides by looking for sugar oxonium ions during intermittent high‐collision energy surveys. Candidate ions are then selected for MSMS, which reveals both the carbohydrate content and the glycosylation site location.Results Glycopeptides were located and site specifically analysed. Data suggests N‐linked occupancy of numerous sites, and microheterogeneity was observed.Discussion This is the first study to investigate the N‐glycans on oligomeric gel‐forming mucins. The N‐glycans are localized in the N‐ and C‐terminal portions of the MUC5B polypeptide in areas which have sparse O‐glycosylation. These regions of the molecule are known to be involved in the disulfide bond‐mediated oligomerization of the mucin monomers. Furthermore, they may be potential sites for interaction with other components of saliva that are important for biological function. It may be that they have a role in stabilizing these important regions of the molecule, or they may be sites of interaction with other salivary components.