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


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 



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.


  1. 1.
    Hassa PO, Haenni SS, Elser M, Hottiger MO (2006) Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going? Microbiol Mol Biol Rev: MMBR 70(3):789–829. doi: 10.1128/MMBR.00040-05 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hottiger MO, Hassa PO, Luscher B, Schuler H, Koch-Nolte F (2010) Toward a unified nomenclature for mammalian ADP-ribosyltransferases. Trends Biochem Sci 35(4):208–219. doi:  10.1016/j.tibs.2009.12.003 CrossRefPubMedGoogle Scholar
  3. 3.
    Vyas S, Matic I, Uchima L, Rood J, Zaja R, Hay RT, Ahel I, Chang P (2014) Family-wide analysis of poly(ADP-ribose) polymerase activity. Nat Commun 5:4426. doi: 10.1038/ncomms5426 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Slade D, Dunstan MS, Barkauskaite E, Weston R, Lafite P, Dixon N, Ahel M, Leys D, Ahel I (2011) The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature 477(7366):616–620. doi: 10.1038/nature10404 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Rosenthal F, Feijs KL, Frugier E, Bonalli M, Forst AH, Imhof R, Winkler HC, Fischer D, Caflisch A, Hassa PO, Luscher B, Hottiger MO (2013) Macrodomain-containing proteins are new mono-ADP-ribosylhydrolases. Nat Struct Mol Biol 20(4):502–507. doi: 10.1038/nsmb.2521 CrossRefPubMedGoogle Scholar
  6. 6.
    Jankevicius G, Hassler M, Golia B, Rybin V, Zacharias M, Timinszky G, Ladurner AG (2013) A family of macrodomain proteins reverses cellular mono-ADP-ribosylation. Nat Struct Mol Biol 20(4):508–514. doi: 10.1038/nsmb.2523 CrossRefPubMedGoogle Scholar
  7. 7.
    Daniels CM, Ong SE, Leung AK (2015) The promise of proteomics for the study of ADP-ribosylation. Mol Cell 58(6):911–924. doi: 10.1016/j.molcel.2015.06.012 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhang Y, Wang J, Ding M, Yu Y (2013) Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome. Nat Methods 10(10):981–984. doi: 10.1038/nmeth.2603 CrossRefPubMedGoogle Scholar
  9. 9.
    Rosenthal F, Messner S, Roschitzki B, Gehrig P, Nanni P, Hottiger MO (2011) Identification of distinct amino acids as ADP-ribose acceptor sites by mass spectrometry. Methods Mol Biol 780:57–66. doi: 10.1007/978-1-61779-270-0_4 CrossRefPubMedGoogle Scholar
  10. 10.
    Gagne JP, Ethier C, Defoy D, Bourassa S, Langelier MF, Riccio AA, Pascal JM, Moon KM, Foster LJ, Ning Z, Figeys D, Droit A, Poirier GG (2015) Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs. DNA Repair 30:68–79. doi: 10.1016/j.dnarep.2015.02.004 CrossRefPubMedGoogle Scholar
  11. 11.
    Matic I, Ahel I, Hay RT (2012) Reanalysis of phosphoproteomics data uncovers ADP-ribosylation sites. Nat Methods 9(8):771–772. doi: 10.1038/nmeth.2106 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lang AE, Schmidt G, Schlosser A, Hey TD, Larrinua IM, Sheets JJ, Mannherz HG, Aktories K (2010) Photorhabdus luminescens toxins ADP-ribosylate actin and RhoA to force actin clustering. Science 327(5969):1139–1142. doi: 10.1126/science.1184557 CrossRefPubMedGoogle Scholar
  13. 13.
    Chapman JD, Gagne JP, Poirier GG, Goodlett DR (2013) Mapping PARP-1 auto-ADP-ribosylation sites by liquid chromatography-tandem mass spectrometry. J Proteome Res 12(4):1868–1880. doi: 10.1021/pr301219h CrossRefPubMedGoogle Scholar
  14. 14.
    Daniels CM, Ong SE, Leung AK (2014) Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells. J Proteome Res 13(8):3510–3522. doi: 10.1021/pr401032q CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Martello R, Leutert M, Jungmichel S, Bilan V, Larsen SC, Young C, Hottiger MO, Nielsen ML (2016) Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue. Nat Commun 7:12917. in pressCrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zhou H, Ye M, Dong J, Han G, Jiang X, Wu R, Zou H (2008) Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis. J Proteome Res 7(9):3957–3967. doi: 10.1021/pr800223m CrossRefPubMedGoogle Scholar
  17. 17.
    Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362. doi: 10.1038/nmeth.1322 CrossRefPubMedGoogle Scholar
  18. 18.
    Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906. doi: 10.1038/nprot.2007.261 CrossRefPubMedGoogle Scholar
  19. 19.
    Rosenthal F, Nanni P, Barkow-Oesterreicher S, Hottiger MO (2015) Optimization of LTQ-orbitrap mass spectrometer parameters for the identification of ADP-ribosylation sites. J Proteome Res 14(9):4072–4079. doi: 10.1021/acs.jproteome.5b00432 CrossRefPubMedGoogle Scholar
  20. 20.
    Hengel SM, Shaffer SA, Nunn BL, Goodlett DR (2009) Tandem mass spectrometry investigation of ADP-ribosylated kemptide. J Am Soc Mass Spectrom 20(3):477–483. doi: 10.1016/j.jasms.2008.10.025 CrossRefPubMedGoogle Scholar
  21. 21.
    Savitski MM, Mathieson T, Becher I, Bantscheff M (2010) H-score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples. J Proteome Res 9(11):5511–5516. doi: 10.1021/pr1006813 CrossRefPubMedGoogle Scholar
  22. 22.
    Daniels CM, Thirawatananond P, Ong SE, Gabelli SB, Leung AK (2015) Nudix hydrolases degrade protein-conjugated ADP-ribose. Sci Rep 5:18271. doi: 10.1038/srep18271 CrossRefPubMedPubMedCentralGoogle Scholar

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
    Email author
  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

Personalised recommendations