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Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography–Tandem Mass Spectrometry

  • Sara C. Larsen
  • Mario Leutert
  • Vera Bilan
  • Rita Martello
  • Stephanie Jungmichel
  • Clifford Young
  • Michael O. Hottiger
  • Michael L. NielsenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1608)

Abstract

ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography–high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.

Key words

ADP-ribosylation ADP-ribosylome Mass spectrometry Proteomics Af1521 macrodomain enrichment Affinity purification PARG 

Notes

Acknowledgments

Ms. Monika Fey is acknowledged for the expression and purification of recombinant human PARG (University of Zurich) and Paolo Nanni for technical support for the MS measurements (FGCZ, University of Zurich). We also thank Felix R. Althaus (Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse) for providing hPARG expressing baculo virus. Stephan Christen and Deena Leslie Petrioli provided editorial assistance and critical input during the writing (University of Zurich). The work carried out in the laboratory of MLN was in part supported by the Novo Nordisk Foundation Center for Protein Research; the Novo Nordisk Foundation (grant number NNF14CC0001 and NNF13OC0006477); the Lundbeck Foundation (Grant number R171-2014-1496); The Danish Council of Independent Research, grant agreement number DFF 4002-00051 (Sapere Aude) and grant agreement number DFF 4183-00322A. ADP-ribosylation research in the laboratory of MOH is funded by the Kanton of Zurich, the University Research Priority Program (URPP) in Translational Cancer Biology at the University of Zurich, and the Swiss National Science Foundation (grant 310030B_138667).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Sara C. Larsen
    • 1
  • Mario Leutert
    • 2
    • 3
  • Vera Bilan
    • 2
    • 3
  • Rita Martello
    • 1
  • Stephanie Jungmichel
    • 1
  • Clifford Young
    • 1
  • Michael O. Hottiger
    • 2
  • Michael L. Nielsen
    • 1
    Email author
  1. 1.Department of Proteomics, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein ResearchUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of Molecular Mechanisms of DiseaseUniversity of ZurichZurichSwitzerland
  3. 3.Molecular Life Science PhD Program of the Life Science Zurich Graduate SchoolUniversity of Zurich/ETH ZurichZurichSwitzerland

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