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

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.