In Vitro Techniques for ADP-Ribosylated Substrate Identification
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.
Key wordsADP-ribosylation Macro domain Af1521 macro-domain purification DMP cross-linker Macro-domain-based pulldown PARP ART ADP-ribosylated substrates ADP-ribose PAR
This study was supported by the Italian Association for Cancer Research (AIRC, Milan, Italy, IG10341 and 14675 to D.C.), TRansforming IDEas in Oncological research award (TRIDEO, AIRC-Fondazione Cariplo, Milan, Italy, IG17524 to C.V.), the PNR-CNR Aging Program, the Flag project Nanomax, and the POR project OcKey. G.G. received a fellowship from the Italian Foundation for Cancer Research (FIRC, Milan, Italy).