Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography–Tandem Mass Spectrometry
Protein ADP-ribosylation is a covalent, reversible posttranslational modification (PTM) catalyzed by ADP-ribosyltransferases (ARTs). Proteins can be either mono- or poly-ADP-ribosylated under a variety of physiological and pathological conditions. To understand the functional contribution of protein ADP-ribosylation to normal and disease/stress states, modified protein and corresponding ADP-ribose acceptor site identification is crucial. Since ADP-ribosylation is a transient and relatively low abundant PTM, systematic and accurate identification of ADP-ribose acceptor sites has only recently become feasible. This is due to the development of specific ADP-ribosylated protein/peptide enrichment methodologies, as well as technical advances in high-accuracy liquid chromatography–tandem mass spectrometry (LC-MS/MS). The standardized protocol described here allows the identification of ADP-ribose acceptor sites in in vitro ADP-ribosylated proteins and will, thus, contribute to the functional characterization of this important PTM.
Key wordsADP-ribosylation ADP-ribosylome ARTD PARP PARG Mass spectrometry Ti4+-IMAC enrichment Phosphoenrichment
The authors would like to thank Paolo Nanni (member of the Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Zurich, Switzerland) for advice and technical assistance. We also thank Felix R. Althaus (Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse) for providing hPARG-expressing baculovirus. Stephan Christen and Deena Leslie Pedrioli (both University of Zurich) provided editorial assistance and critical input during the writing. Work on ADP-ribosyltransferases in the laboratory of M.O.H is supported by Kanton of Zurich and the Swiss National Science Foundation (310030_157019).
Mario Leutert and Vera Bilan contributed equally to this chapter.
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