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)

    Research output: Contribution to journalArticle

    115 Citations (Scopus)
    115 Downloads (Pure)

    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

    Fingerprint

    enzyme activity
    regulators
    Ubiquitin
    linkages
    inhibitors
    Ionization
    Mass spectrometry
    Assays
    Desorption
    Mass Spectrometry
    Screening
    drugs
    Lasers
    mass spectroscopy
    screening
    selectivity
    desorption
    Polyubiquitin
    proteins
    ionization

    Cite this

    Ritorto, M. S., Ewan, R., Perez-Oliva, A. B., Knebel, A., Buhrlage, S. J., Wightman, M., ... Trost, M. (2014). Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nature Communications, 5, [4763]. https://doi.org/10.1038/ncomms5763
    Ritorto, Maria Stella ; Ewan, Richard ; Perez-Oliva, Ana B. ; Knebel, Axel ; Buhrlage, Sara J. ; Wightman, Melanie ; Kelly, Sharon M. ; Wood, Nicola T. ; Virdee, Satpal ; Gray, Nathanael S. ; Morrice, Nicholas A. ; Alessi, Dario R. ; Trost, Matthias. / Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. In: Nature Communications. 2014 ; Vol. 5.
    @article{185d4e1fd918425fbe32fa3b4b47e9da,
    title = "Screening of DUB activity and specificity by MALDI-TOF mass spectrometry",
    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.",
    author = "Ritorto, {Maria Stella} and Richard Ewan and Perez-Oliva, {Ana B.} and Axel Knebel and Buhrlage, {Sara J.} and Melanie Wightman and Kelly, {Sharon M.} and Wood, {Nicola T.} and Satpal Virdee and Gray, {Nathanael S.} and Morrice, {Nicholas A.} and Alessi, {Dario R.} and Matthias Trost",
    year = "2014",
    month = "8",
    day = "27",
    doi = "10.1038/ncomms5763",
    language = "English",
    volume = "5",
    journal = "Nature Communications",
    issn = "2041-1723",
    publisher = "Nature Publishing Group",

    }

    Ritorto, MS, Ewan, R, Perez-Oliva, AB, Knebel, A, Buhrlage, SJ, Wightman, M, Kelly, SM, Wood, NT, Virdee, S, Gray, NS, Morrice, NA, Alessi, DR & Trost, M 2014, 'Screening of DUB activity and specificity by MALDI-TOF mass spectrometry', Nature Communications, vol. 5, 4763. https://doi.org/10.1038/ncomms5763

    Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. / Ritorto, Maria Stella; Ewan, Richard; Perez-Oliva, Ana B.; Knebel, Axel; Buhrlage, Sara J.; Wightman, Melanie; Kelly, Sharon M.; Wood, Nicola T.; Virdee, Satpal; Gray, Nathanael S.; Morrice, Nicholas A.; Alessi, Dario R.; Trost, Matthias (Lead / Corresponding author).

    In: Nature Communications, Vol. 5, 4763, 27.08.2014.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

    AU - Ritorto, Maria Stella

    AU - Ewan, Richard

    AU - Perez-Oliva, Ana B.

    AU - Knebel, Axel

    AU - Buhrlage, Sara J.

    AU - Wightman, Melanie

    AU - Kelly, Sharon M.

    AU - Wood, Nicola T.

    AU - Virdee, Satpal

    AU - Gray, Nathanael S.

    AU - Morrice, Nicholas A.

    AU - Alessi, Dario R.

    AU - Trost, Matthias

    PY - 2014/8/27

    Y1 - 2014/8/27

    N2 - 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.

    AB - 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.

    U2 - 10.1038/ncomms5763

    DO - 10.1038/ncomms5763

    M3 - Article

    VL - 5

    JO - Nature Communications

    JF - Nature Communications

    SN - 2041-1723

    M1 - 4763

    ER -

    Ritorto MS, Ewan R, Perez-Oliva AB, Knebel A, Buhrlage SJ, Wightman M et al. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nature Communications. 2014 Aug 27;5. 4763. https://doi.org/10.1038/ncomms5763