Combining Metabolic Alkyne Labeling and Click Chemistry for Secretome Analysis of Serum-Containing Conditioned Medium†

Main observation and conclusion Bioorthogonal click chemistry has emerged as a powerful tool for the specific modification of proteins in complex mixtures. Metabolic labeling of proteins with azide followed by the copper-catalyzed azide-alkyne cycloaddition (CuAAC) with alkyne-based affinity probes/beads is widely applied to study protein turnover and post-translational modifications (PTMs). However, it has long been known that the alkyne-based enrichment of high concentration protein samples (e.g., serum) is not sufficient to remove high abundant contaminating proteins. Herein, we demonstrate that the protein contamination is mainly caused by thiol-yne addition between terminal alkyne and cysteine residue. Furthermore, we report that azide-based enrichment combined with metabolic labeling with alkyne can significantly reduce contamination and improve the enrichment efficiency of secreted proteins in serum-containing conditioned medium.