Skip to main content
SpringerLink
Log in

Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry

  • Protocol
  • First Online:
Poly(ADP-Ribose) Polymerase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2609))

Abstract

ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911–1920, 2005), followed by liquid chromatography–high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Abplanalp J, Leutert M, Frugier E, Nowak K, Feurer R, Kato J, Kistemaker HVA, Filippov DV, Moss J, Caflisch A, Hottiger MO (2017) Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase. Nat Commun 8(1). https://doi.org/10.1038/s41467-017-02253-1

  2. Altmeyer M, Messner S, Hassa PO, Fey M, Hottiger MO (2009) Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites. Nucleic Acids Res 37(11):3723–3738. https://doi.org/10.1093/nar/gkp229

    Article  CAS  Google Scholar 

  3. Amé J, Héberlé É, Camuzeaux B, Dantzer F, Schreiber V (2017) Purification of recombinant human PARG and activity assays. Methods Mol Biol 1608(1):395–413. https://doi.org/10.1007/978-1-4939-6993-7

    Article  Google Scholar 

  4. Bilan V, Leutert M, Nanni P, Panse C, Hottiger MO (2017) Combining higher-energy collision dissociation and electron-transfer/higher-energy collision dissociation fragmentation in a product-dependent manner confidently assigns proteomewide ADP-ribose acceptor sites. Anal Chem 89(3):1523–1530. https://doi.org/10.1021/acs.analchem.6b03365

    Article  CAS  Google Scholar 

  5. Bonfiglio JJ, Colby T, Matic I (2017a) Mass spectrometry for serine ADP-ribosylation? Think o-glycosylation! Nucleic Acids Res 45(11):6259–6264. https://doi.org/10.1093/nar/gkx446

    Article  CAS  Google Scholar 

  6. Bonfiglio JJ, Fontana P, Zhang Q, Colby T, Gibbs-Seymour I, Atanassov I, Bartlett E, Zaja R, Ahel I, Matic I (2017b) Serine ADP-ribosylation depends on HPF1. Mol Cell 65(5):932–940.e6. https://doi.org/10.1016/j.molcel.2017.01.003

    Article  CAS  Google Scholar 

  7. Buch-Larsen SC, Hendriks IA, Lodge JM, Rykær M, Furtwängler B, Shishkova E, Westphall MS, Coon JJ, Nielsen ML (2020) Mapping physiological ADP-ribosylation using activated ion electron transfer dissociation. Cell Rep 32(12). https://doi.org/10.1016/j.celrep.2020.108176

  8. Buch-Larsen SC, Rebak AKLFS, Hendriks IA, Nielsen ML (2021) Temporal and site-specific adp-ribosylation dynamics upon different genotoxic stresses. Cell 10(11). https://doi.org/10.3390/cells10112927

  9. Cervantes-Laurean D, Loflin PT, Mintei DE, Jacobsonll EL, Jacobson MK (1995) Protein modification by ADP-ribose via acid-labile linkages. J Biol Chem 270(14):7929–7936. https://doi.org/10.1074/jbc.270.14.7929

    Article  CAS  Google Scholar 

  10. Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325(5942):834–840. https://doi.org/10.1126/science.1175371

    Article  CAS  Google Scholar 

  11. Cohen MS, Chang P (2018) Insights into the biogenesis, function, and regulation of ADP-ribosylation. Nat Chem Biol 14(3):236–243. https://doi.org/10.1038/nchembio.2568

    Article  CAS  Google Scholar 

  12. Hendriks IA, Buch-Larsen SC, Prokhorova E, Elsborg JD, Rebak AKLFS, Zhu K, Ahel D, Lukas C, Ahel I, Nielsen ML (2021) The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome. Nat Commun 12(1). https://doi.org/10.1038/s41467-021-26172-4

  13. Hendriks IA, Larsen SC, Nielsen ML (2019) An advanced strategy for comprehensive profiling of ADP-ribosylation sites using mass spectrometry-based proteomics. Mol Cell Proteomics 18(5):1010–1024. https://doi.org/10.1074/mcp.TIR119.001315

    Article  CAS  Google Scholar 

  14. Hendriks IA, Souza RCJD, Yang B, Verlaan-De Vries M, Mann M, Vertegaal ACO (2014) Uncovering global SUMOylation signaling networks in a site-specific manner. Nat Publ Group. https://doi.org/10.1038/nsmb.2890

  15. Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG (2005) The macro domain is an ADP-ribose binding module. EMBO J 24(11):1911–1920. https://doi.org/10.1038/sj.emboj.7600664

    Article  CAS  Google Scholar 

  16. Larsen SC, Hendriks IA, Lyon D, Jensen LJ, Nielsen ML (2018a) Systems-wide analysis of serine ADP-ribosylation reveals widespread occurrence and site-specific overlap with phosphorylation. Cell Rep 24(9):2493–2505.e4. https://doi.org/10.1016/j.celrep.2018.07.083

    Article  CAS  Google Scholar 

  17. Larsen SC, Sylvestersen KB, Mund A, Lyon D, Mullari M, Madsen MV, Daniel JA, Jensen LJ, Nielsen ML (2016) Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells. Sci Signal 9(443). https://doi.org/10.1126/scisignal.aaf7329

  18. Larsen S, Leutert M, Bilan V, Martello R, Jungmichel S, Young C, Hottiger MO, Nielsen ML (2018b) Proteome-wide identification of in vivo ADP-ribose acceptor sites by liquid chromatography–tandem mass spectrometry. Methods Mol Biol 1608(1):231–253. https://doi.org/10.1007/978-1-4939-6993-7

    Article  Google Scholar 

  19. Leutert M, Menzel S, Braren R, Rissiek B, Hopp AK, Nowak K, Bisceglie L, Gehrig P, Li H, Zolkiewska A, Koch-Nolte F, Hottiger MO (2018) Proteomic characterization of the heart and skeletal muscle reveals widespread arginine ADP-ribosylation by the ARTC1 ectoenzyme. Cell Rep 24(7):1916–1929.e5. https://doi.org/10.1016/j.celrep.2018.07.048

    Article  CAS  Google Scholar 

  20. Lüscher B, Ahel I, Altmeyer M, Ashworth A, Bai P, Chang P, Cohen M, Corda D, Dantzer F, Daugherty MD, Dawson TM (2021) ADP‐ribosyltransferases, an update on function and nomenclature. The FEBS Journal. https://doi.org/10.1111/febs.16142

  21. 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 7:1–13. https://doi.org/10.1038/ncomms12917

    Article  CAS  Google Scholar 

  22. Mcdonald LJ, Moss J (1994) Enzymatic and nonenzymatic ADP-ribosylation of cysteine. In: Molecular and cellular biochemistry, vol 138

    Google Scholar 

  23. Moss J, Vaughan M (1977) Mechanism of action of choleragen. Evidence for ADP ribosyltransferase activity with arginine as an acceptor. J Biol Chem 252(7):2455–2457. https://doi.org/10.1016/s0021-9258(17)40578-3

    Article  CAS  Google Scholar 

  24. Ogata N, Ueda K, Hayaishi O (1980) ADP-ribosylation of histone H2B. Identification of glutamic acid residue 2 as the modification site. J Biol Chem 255(16):7610–7615. https://doi.org/10.1016/S0021-9258(19)43872-6

    Article  CAS  Google Scholar 

  25. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127(3):635–648. https://doi.org/10.1016/J.CELL.2006.09.026

    Article  CAS  Google Scholar 

  26. Oppenheimer NJ, Bodley JW (1981) Diphtheria toxin. Site and configuration of ADP-ribosylation of diphthamide in elongation factor 2. J Biol Chem 256(16):8579–8581. https://doi.org/10.1016/S0021-9258(19)68883-6

    Article  CAS  Google Scholar 

  27. Palazzo L, Leidecker O, Prokhorova E, Dauben H, Matic I, Ahel I (2018) Serine is the major residue for ADP-ribosylation upon DNA damage. elife 7:1–12. https://doi.org/10.7554/eLife.34334

    Article  Google Scholar 

  28. 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):1896-906. https://doi.org/10.1038/nprot.2007.261

  29. Rodriguez KM, Buch-Larsen SC, Kirby IT, Siordia IR, Hutin D, Rasmussen M, Grant DM, David LL, Matthews J, Nielsen ML, Cohen MS (2021) Chemical genetics and proteome-wide site mapping reveal cysteine MARylation by PARP-7 on immune-relevant protein targets. elife 10. https://doi.org/10.7554/eLife.60480

  30. 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. https://doi.org/10.1038/nmeth.2603

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Proteomics program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Holda A. Anagho, Jonas D. Elsborg, Ivo A. Hendriks, Sara C. Buch-Larsen & Michael L. Nielsen

Authors
  1. Holda A. Anagho
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jonas D. Elsborg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ivo A. Hendriks
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sara C. Buch-Larsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael L. Nielsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael L. Nielsen .

Editor information

Editors and Affiliations

  1. Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA

    Alexei V. Tulin

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Anagho, H.A., Elsborg, J.D., Hendriks, I.A., Buch-Larsen, S.C., Nielsen, M.L. (2023). Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry. In: Tulin, A.V. (eds) Poly(ADP-Ribose) Polymerase. Methods in Molecular Biology, vol 2609. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2891-1_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2891-1_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2890-4

  • Online ISBN: 978-1-0716-2891-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation