In Vitro Techniques for ADP-Ribosylated Substrate Identification

Abstract

ADP-ribosylation is a post-translational modification of proteins that has required the development of specific technical approaches for the full definition of its physiological roles and regulation. The identification of the enzymes and specific substrates of this reaction is an instrumental step toward these aims. Here we describe a method for the separation of ADP-ribosylated proteins based on the use of the ADP-ribose-binding macro domain of the thermophilic protein Af1521, coupled to mass spectrometry analysis for protein identification. This method foresees the coupling of the macro domain to resin, an affinity-based pull-down assay, coupled to a specificity step resulting from the clearing of cell lysates with a mutated macro domain unable to bind ADP-ribose. By this method both mono- and poly-ADP-ribosylated proteins have been identified.

Keywords: ADP-ribose; ADP-ribosylated substrates; ADP-ribosylation; ART; Af1521 macro-domain purification; DMP cross-linker; Macro domain; Macro-domain-based pulldown; PAR; PARP.