Automated High-Throughput Method for the Fast, Robust, and Reproducible Enrichment of Newly Synthesized Proteins

[Image: see text] A high-throughput method was developed for the automated enrichment of newly synthesized proteins (NSPs), which are labeled metabolically by substituting methionine with the “click-able” analogue azidohomoalanine (AHA). A suitable conjugate containing a dibenzocyclooctyne (DBCO) group allows the specific selection of NSPs by a fast 1 h click chemistry-based reaction with AHA. Through an automated pipetting platform, the samples are loaded into streptavidin cartridges for the selective binding of the NSPs by means of a biotin bait contained in the conjugate. The enriched proteins are eluted by a reproducible chemical cleavage of the 4,4-dimethyl-2,6-dioxocyclohexylidene (Dde) group in the conjugate, which increases selectivity. The NSPs can be collected and digested in the same well plate, and the resulting peptides can be subsequently loaded for automated cleanup, followed by mass spectrometry analysis. The proposed automated method allows for the robust and effective enrichment of samples in 96-well plates in a period of 3 h. Our developed enrichment method was comprehensively evaluated and then applied to the proteomics analysis of the melanoma A375 cell secretome, after treatment with the cytokines interferon α (IFN-α) and γ (IFN-γ), resulting in the quantification of 283 and 263 proteins, respectively, revealing intricate tumor growth-supportive and -suppressive effects.