| Summary: | Bovine submaxillary mucin (BSM) is a gel-forming glycoprotein polymer, and Ser/Thr-linked glycans (<i>O</i>-glycans) are important in regulating BSM’s viscoelasticity and polymerization. However, details of <i>O</i>-glycosylation have not been reported. This study investigates the structural and quantitative characteristics of <i>O</i>-glycans and identifies <i>O</i>-glycosylation sites in BSM using liquid chromatography–tandem mass spectrometry. The <i>O</i>-glycans (consisting of di- to octa-saccharides) and their quantities (%) relative to total <i>O</i>-glycans (100%; 1.1 pmol per 1 μg of BSM) were identified with 14 major (>1.0%), 12 minor (0.1%–1.0%), and eight trace (<0.1%) <i>O</i>-glycans, which were characterized based on their constituents (sialylation (14 <i>O</i>-glycans; 81.9%, sum of relative quantities of each glycan), non-sialylation (20; 18.1%), fucosylation (20; 17.5%), and terminal-galactosylation (6; 3.6%)) and six core structure types [Gal-GalNAc, Gal-(GlcNAc)GalNAc, GlcNAc-GalNAc, GlcNAc-(GlcNAc)GalNAc, and GalNAc-GalNAc]. <i>O</i>-glycosylation sites were identified using <i>O</i>-glycopeptides (bold underlined; <sub>56</sub><b>S</b>GE<b>T</b>R<b>TS</b>VI, <sub>259</sub><b>S</b>H<b>SSS</b>GR<b>S</b>R<b>T</b>I, <sub>272</sub>G<b>S</b>P<b>SS</b>V<b>SS</b>AEQI, <sub>307</sub>RP<b>S</b>YGAL, <sub>625</sub>Q<b>T</b>LGPL, <sub>728</sub><b>T</b>M<b>TT</b>R<b>TS</b>VVV, and <sub>1080</sub>RPEDN<b>T</b>AVA) obtained from proteolytic BSM; these sites are in the four domains of BSM. The gel-forming mucins share common domain structures and glycosylation patterns; these results could provide useful information on mucin-type <i>O</i>-glycans. This is the first study to characterize <i>O</i>-glycans and identify <i>O</i>-glycosylation sites in BSM.
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