Despite substantial progress in ADP-ribosylation research in recent years, the identification of ADP-ribosylated proteins, their ADP-ribose acceptors sites, and the respective writers and erasers remains challenging. The use of recently developed mass spectrometric methods helps to further characterize the ADP-ribosylome and its regulatory enzymes under different conditions and in different cell types. Validation of these findings may be achieved by in vitro assays for the respective enzymes. In the below method, we describe how recombinant ADP-ribosylated proteins are demodified in vitro with mono-ADP-ribosylhydrolases of choice to elucidate substrate and potentially also site specificity of these enzymes.
Key wordsMono-ADP-ribosylhydrolases Macrodomain De-ADP-ribosylation assay PARG MACROD2 MACROD1 ARH1 ARH3 TARG OARD1 C6ORF130
The authors would like to thank Tobias Suter (University of Zurich) for providing editorial assistance and critical input during manuscript writing. Work on ADP-ribosyltransferases and hydrolases in the laboratory of MOH is supported by the Kanton of Zurich and the Swiss National Science Foundation (SNF 310030_157019 and 31003A_176177).
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