Fig. 4.

Schematic representation of the enrichment of secreted proteins by metabolic-labeling with azido sugars using the hiSPECS approach. ① Protein secretion is probed by collecting cell culture supernatants from ManNAz-labeled cells. ② Glycoproteins are first enriched from the serum-containing cell culture medium via a lectin-based glycoprotein enrichment using concanavalin A beads. Beads are washed and the glycoproteins are eluted through competition with methyl-alpha-D-mannopyranoside. The azido-glycoproteins are then selectively captured from the bulk of serum-derived glycoproteins via covalent binding to magnetic dibenzylcyclooctyne (DBCO)–alkyne beads through copper-free click chemistry, which enables a stringent wash to remove unspecific bound serum proteins. ③ Bead-bound proteins are digested and the resulting peptide samples are ④ analyzed by LC-MS/MS. hiSPECS, high-performance secretome enrichment with click sugars.