Discovery of Electrophiles and Profiling of Enzyme Cofactors

Suzanne E. Dettling, Mina Ahmadi, Zongtao Lin, Lin He, Megan L. Matthews

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Reverse-polarity activity-based protein profiling (RP-ABPP) is a chemical proteomics approach that uses nucleophilic probes amenable to “click” chemistry deployed into living cells in culture to capture, immunoprecipitate, and identify protein-bound electrophiles. RP-ABPP is used to characterize the structure and function of reactive electrophilic post-translational modifications (PTMs) and the proteins harboring them, which may uncover unknown or novel functions. RP-ABPP has demonstrated utility as a versatile method to monitor the metabolic regulation of electrophilic cofactors, using a pyruvoyl cofactor in S-adenosyl-L-methionine decarboxylase (AMD1), and to discover novel types of electrophilic modifications on proteins in human cells, such as the glyoxylyl modification on secernin-3 (SCRN3). These cofactors cannot be predicted by sequence, and therefore this area is relatively undeveloped. RP-ABPP is the only global, unbiased approach to discover such electrophiles. Here, we describe the utility of these experiments and provide a detailed protocol for de novo discovery, quantitation, and global profiling of electrophilic functionality of proteins.

Original languageEnglish
Article numbere86
JournalCurrent protocols in chemical biology
Volume12
Issue number4
DOIs
StatePublished - Dec 2020

Keywords

  • chemoproteomics
  • de novo PTM discovery
  • enzyme activity
  • post-translational modifications
  • reverse-polarity activity-based protein profiling

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