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.