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

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. Morrice, Dario R. Alessi, Matthias Trost (Lead / Corresponding author)

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    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.
    Original languageEnglish
    Article number4763
    Number of pages11
    JournalNature Communications
    Volume5
    DOIs
    Publication statusPublished - 27 Aug 2014

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    Cite this

    Ritorto, M. S., Ewan, R., Perez-Oliva, A. B., Knebel, A., Buhrlage, S. J., Wightman, M., Kelly, S. M., Wood, N. T., Virdee, S., Gray, N. S., Morrice, N. A., Alessi, D. R., & Trost, M. (2014). Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nature Communications, 5, [4763]. https://doi.org/10.1038/ncomms5763