Poly(ADP-Ribose) Polymerase pp 137-148

Part of the Methods in Molecular Biology book series (MIMB, volume 1608)

Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography–Tandem Mass Spectrometry

  • Mario Leutert
  • Vera Bilan
  • Peter Gehrig
  • Michael O. Hottiger
Protocol

Abstract

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 words

ADP-ribosylation ADP-ribosylome ARTD PARP PARG Mass spectrometry Ti4+-IMAC enrichment Phosphoenrichment 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Mario Leutert
    • 1
    • 2
  • Vera Bilan
    • 1
    • 2
  • Peter Gehrig
    • 3
  • Michael O. Hottiger
    • 1
  1. 1.Department of Molecular Mechanisms of DiseaseUniversity of ZurichZurichSwitzerland
  2. 2.Molecular Life Science PhD Program of the Life Science Zurich Graduate SchoolUniversity of Zurich/ETH ZurichZurichSwitzerland
  3. 3.Functional Genomics Center ZurichUniversity of Zurich/ETH ZurichZurichSwitzerland

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