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

doi:10.1038/ncomms5763

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 language	English
Article number	4763
Number of pages	11
Journal	Nature Communications
Volume	5
DOIs	https://doi.org/10.1038/ncomms5763
Publication status	Published - 27 Aug 2014

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A Systematic Investigation into the Pathogenesis and Course of Parkinson's Syndrome (Wellcome Trust and MRC Neurodegenerative Diseases Initiative) (Joint with University College London)
Alessi, D. (Investigator)
Medical Research Council
1/09/10 → 31/08/15
Project: Research

Alessi, Dario
Person: Academic

Cite this

@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 = aug,

day = "27",

doi = "10.1038/ncomms5763",

language = "English",

volume = "5",

journal = "Nature Communications",

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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.

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SN - 2041-1723

VL - 5

JO - Nature Communications

JF - Nature Communications

M1 - 4763

ER -

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

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A Systematic Investigation into the Pathogenesis and Course of Parkinson's Syndrome (Wellcome Trust and MRC Neurodegenerative Diseases Initiative) (Joint with University College London)

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Alessi, Dario

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