Abstract
Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability. N-glycans are important for mucin stability and function, but their structures have not been fully characterized, unlike that of O-glycans. In this study, BSM N-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32 N-glycans were identified, and the quantities (%) of each N-glycan relative to total N-glycans (100%) were obtained. The terminal N-acetylgalactosamines in 12 N-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated N-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present N-glycans and reported O-glycans). No N-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM N-glycans. Mucin structures, including sulfated N-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated N-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.
Highlights
Asparagine-linked (N-) glycans containing negatively charged moieties, such as sialic acid, phosphate, or sulfate groups, frequently have crucial roles in the function of glycoproteins [1]
The N-glycans released from Bovine submaxillary mucin (BSM) were derivatized with ProA and analyzed using ultraperformance liquid chromatography (UPLC) and liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD)-mass spectrometry (MS)/MS
N-glycan peaks were obtained from UPLC chromatograms (Figure 1A) and the total ion chromatogram (TIC; Figure 1D) corresponding to 32 N-glycan structures (Figure S1), which were identified by the extracted-ion chromatogram (EIC) of two diagnostic oxonium ions: [Sulfate(-SO4;S)1 N-acetylhexosamine(HexNAc)1 + H]+ and [ProAFuc1HexNAc1 + H]+
Summary
Asparagine-linked (N-) glycans containing negatively charged moieties, such as sialic acid, phosphate, or sulfate groups, frequently have crucial roles in the function of glycoproteins [1]. Phosphorylated N-glycans, including the mannose-6-phosphate group, are responsible for the efficacy of enzyme replacement therapy [3], and sulfated N-glycans are essential for electrostatic interactions and signal transduction pathways [4]. These N-glycans are difficult to analyze because of the labile nature of their charged moiety, their heterogeneous composition and structure, and their small proportions relative to total glycan quantities [5,6]. AB is the most frequently used fluorophore for the labeling of glycans [8,9]; ProA-labeling improves the ability to identify glycans using electrospray ionization (ESI)-MS [10], enhances MS ionization efficiency approximately 10–50 times [9], and minimizes the ionization deviation of charged glycans by increasing hydrophobicity [11]
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