Fig. 3.

Schematic representation of the enrichment of secreted proteins using a dual labeling approach with azidohomoalanine in combination with stable isotope labeling by amino acids in cell culture. ① To analyze proteins in serum-containing media, a pulse labeling with AHA in combination with SILAC is performed. ② Newly synthesized proteins carrying the AHA label and that are secreted into the cell culture supernatant are enriched through covalent coupling to an alkyne-activated agarose resin via a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC-mediated click chemistry). The covalent coupling to the agarose resin allows stringent washing for the removal of high abundant serum proteins and other contaminants. ③ Resin-bound proteins are digested and the resulting peptide samples are ④ analyzed by LC-MS/MS. AHA, azidohomoalanine; SILAC, stable isotope labeling with amino acids in cell culture.