A Simplified and Ultrafast Pipeline for Site-Specific Quantitative Chemical Proteomics

Activity-based proteome profiling (ABPP) is a powerful
chemoproteomic
technology for global profiling of protein activity and modifications.
The tandem orthogonal proteolysis-ABPP (TOP-ABPP) strategy utilizes
a clickable enrichment tag with cleavable linkers to enable direct
identification of probe-labeled residue sites within the target proteins.
However, such a site-specific chemoproteomic workflow requires a long
operation time and complex sample preparation procedures, limiting
its wide applications. In the current study, we developed a simplified
and ultrafast peptide enrichment and release TOP-ABPP (“superTOP-ABPP”)
pipeline for site-specific quantitative chemoproteomic analysis with
special agarose resins that are functionalized with azide groups and
acid-cleavable linkers. The azide groups allow enrichment of peptides
that are labeled by the alkynyl probe through a one-step click reaction,
which can be conveniently released by acid cleavage for subsequent
LC-MS/MS analysis. In comparison with the traditional TOP-ABPP method,
superTOP-ABPP cuts down the averaged sample preparation time from
25 to 9 h, and significantly improves the sensitivity and coverage
of site-specific cysteinome profiling. The method can also be seamlessly
integrated with reductive dimethylation to enable quantitative chemoproteomic
analysis with a high accuracy. The simplified and ultrafast superTOP-ABPP
will become a valuable tool for site-specific quantitative chemoproteomic
studies.