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Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Author

Listed:
  • Maria Stella Ritorto

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Richard Ewan

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Ana B. Perez-Oliva

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Axel Knebel

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Sara J. Buhrlage

    (Dana-Farber Cancer Institute
    Harvard Medical School)

  • Melanie Wightman

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Sharon M. Kelly

    (Institute of Molecular Cell and Systems Biology, University of Glasgow)

  • Nicola T. Wood

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Satpal Virdee

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Nathanael S. Gray

    (Dana-Farber Cancer Institute
    Harvard Medical School)

  • Nicholas A. Morrice

    (The Beatson Institute for Cancer Research, Bearsden)

  • Dario R. Alessi

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

  • Matthias Trost

    (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee)

Abstract

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analysing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAB/MPN/Mov34 metalloenzyme DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.

Suggested Citation

  • Maria Stella Ritorto & Richard Ewan & Ana B. Perez-Oliva & Axel Knebel & Sara J. Buhrlage & Melanie Wightman & Sharon M. Kelly & Nicola T. Wood & Satpal Virdee & Nathanael S. Gray & Nicholas A. Morric, 2014. "Screening of DUB activity and specificity by MALDI-TOF mass spectrometry," Nature Communications, Nature, vol. 5(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:5:y:2014:i:1:d:10.1038_ncomms5763
    DOI: 10.1038/ncomms5763
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