
Discovery of Electrophiles and Profiling of Enzyme Cofactors
Author(s) -
Dettling Suzanne E.,
Ahmadi Mina,
Lin Zongtao,
He Lin,
Matthews Megan L.
Publication year - 2020
Publication title -
current protocols in chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.503
H-Index - 14
ISSN - 2160-4762
DOI - 10.1002/cpch.86
Subject(s) - cofactor , profiling (computer programming) , enzyme , electrophile , computational biology , chemistry , biology , biochemistry , computer science , catalysis , operating system
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