Fig. 1

Overview of the in vivo glycocapture method for labelling luminal glycoproteins at the blood–brain barrier. A Depiction of the typical neurovascular unit at the BBB, showing the lumen of the blood vessel and its surrounding cells and basement membrane (BM). B Perfusion-based labelling of glycoproteins in rats and mice using a mild oxidation solution to form aldehydes on luminal glycans, including glycoproteins. C Glycocapture sample preparation workflow to specifically isolate peptides from proteins with oxidized glycans. A hydrazide (Hz) functionalized bead forms a covalent bond with aldehydes, capturing labelled glycoproteins and allowing unbound proteins to be washed away. Bound glycoproteins are then digested with a protease (trypsin, chymotrypsin) and unbound non-glycopeptides are washed away. Formerly N-glycosylated peptides are specifically released from the beads by the enzyme PNGase F and this collection of peptides is analyzed by mass spectrometry. Created with BioRender.com