Discovery of Electrophiles and Profiling of Enzyme Cofactors

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. © 2020 The Authors. Basic Protocol 1: Identification and quantification of probe‐reactive proteins Basic Protocol 2: Characterization of the site of probe labeling Basic Protocol 3: Determination and quantitation of electrophile structure