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A Surface Shave: Revealing the Differentially-Expressed Apical Uroglycome

ABSTRACT: This study aimed to investigate the highly-differentiated urothelial apical surface glycome. The functions of the mammalian urothelium, lining the majority of the urinary tract and providing a barrier against toxins in urine, are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification and complex assembly to regulate the formation of multiple differentiated cell layers. Protein glycosylation, a poorly studied aspect of urothelial differentiation, contributes to the apical glycome and is implicated in the development of urothelial diseases. To enable surface glycome characterization, we have developed a method to collect the tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies was developed for enzymatic shaving of the luminal urothelial surface, with subsequent release and mass spectrometric analysis of apical surface O- and N-glycans; the first normal mammalian urothelial N-glycome to be defined. Trypsinization of superficial glycoproteins was tracked using immunolabelling of the apically-expressed uroplakin 3a protein to optimize enzymatic release, without compromising the integrity of the superficial urothelial layer. The approach developed for releasing apical tissue surface glycans allowed comparison with the N-glycome of total porcine urothelial cells, and thus identification of apical surface glycans as candidates implicated in urothelial barrier function.

INSTRUMENT(S): solariX, Orbitrap Fusion

ORGANISM(S): Pig Bladder Urothelium

SUBMITTER: Edmund Bergstrom Jennifer Southgate Chung-Yao Wang Jane Thomas-Oates

PROVIDER: MSV000087851 | MassIVE | Mon Jul 19 09:25:00 BST 2021

REPOSITORIES: MassIVE

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