A Simplified and Ultrafast Pipeline for Site-specific Quantitative Chemical Proteomics
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ABSTRACT: Activity-based proteome profiling (ABPP) is a powerful chemoproteomic technology for global profiling of protein reactivity 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 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 the alkynyl probe labeled peptides through 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 high accuracy. The simplified and ultrafast superTOP-ABPP will become a valuable tool for site-specific quantitative chemoproteomic studies.
ORGANISM(S): Homo Sapiens
SUBMITTER: Chu Wang
PROVIDER: PXD040880 | iProX | Wed Mar 15 00:00:00 GMT 2023
REPOSITORIES: iProX
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