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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.
Affiliation
  • Ritorto MS; MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
  • Ewan R; 1] MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK [2].
  • Perez-Oliva AB; 1] MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK [2].
  • Knebel A; MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
  • Buhrlage SJ; 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.
  • Wightman M; MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
  • Kelly SM; Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
  • Wood NT; MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
  • Virdee S; MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
  • Gray NS; 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.
  • Morrice NA; The Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, Scotland, UK.
  • Alessi DR; MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
  • Trost M; MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Nat Commun ; 5: 4763, 2014 Aug 27.
Article in En | MEDLINE | ID: mdl-25159004
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.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Protease Inhibitors / Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Ubiquitin-Specific Proteases Type of study: Diagnostic_studies / Screening_studies Limits: Humans Language: En Journal: Nat Commun Journal subject: BIOLOGIA / CIENCIA Year: 2014 Document type: Article Affiliation country:
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Protease Inhibitors / Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Ubiquitin-Specific Proteases Type of study: Diagnostic_studies / Screening_studies Limits: Humans Language: En Journal: Nat Commun Journal subject: BIOLOGIA / CIENCIA Year: 2014 Document type: Article Affiliation country: