Published in

Nature Research, Nature Communications, 1(5), 2014

DOI: 10.1038/ncomms5763

Links

Tools

Export citation

Search in Google Scholar

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

Journal article published in 2014 by Maria Stella Ritorto, Richard Ewan, Ana B. Perez-Oliva, Axel Knebel, Sara J. Buhrlage, Melanie Wightman, Nicola T. Wood ORCID, Ana Perez Oliva, Sharon M. Kelly, Satpal Virdee, Nathanael S. Gray, Nicholas A. Morrice, Dario R. Alessi ORCID, Matthias Trost ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

Full text: Download

Green circle
Preprint: archiving allowed
Upload
Red circle
Postprint: archiving forbidden
Green circle
Published version: archiving allowed
Upload
Policy details
Data provided by SHERPA/RoMEO

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.