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.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Maria Stella
authors.initials: MS
authors.lastname: Ritorto
authors.affiliation: 1] MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK [2].
authors.forename: Richard
authors.initials: R
authors.lastname: Ewan
authors.affiliation: 1] MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK [2].
authors.forename: Ana B
authors.initials: AB
authors.lastname: Perez-Oliva
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.affiliation: 1] Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA [2] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, SGM 628, Boston, Massachusetts 02115, USA.
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Buhrlage
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Melanie
authors.initials: M
authors.lastname: Wightman
authors.affiliation: Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Kelly
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Nicola T
authors.initials: NT
authors.lastname: Wood
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Satpal
authors.initials: S
authors.lastname: Virdee
authors.affiliation: 1] Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA [2] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, SGM 628, Boston, Massachusetts 02115, USA.
authors.forename: Nathanael S
authors.initials: NS
authors.lastname: Gray
authors.affiliation: The Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, Scotland, UK.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Morrice
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Matthias
authors.initials: M
authors.lastname: Trost
mesh_terms: D006801:Humans; D020128:Inhibitory Concentration 50; D009570:Nitriles / Q000494:pharmacology; D009581:Nitrofurans / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity; D013450:Sulfones / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000076084:Ubiquitin-Specific Peptidase 7; D064570:Ubiquitin-Specific Proteases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C000604167:2-((4-methylphenyl)sulfonyl)-5-nitrofuran; C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D009570:Nitriles; D009581:Nitrofurans; D011480:Protease Inhibitors; D013450:Sulfones; D025801:Ubiquitin; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7; D064570:Ubiquitin-Specific Proteases