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High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery

Lookup NU author(s): Professor Matthias TrostORCiD

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Abstract

© 2020, The Author(s), under exclusive licence to Springer Nature Limited.Deubiquitylating enzymes (DUBs) play a vital role in the ubiquitin pathway by editing or removing ubiquitin from their substrate. As breakthroughs within the ubiquitin field continue to highlight the potential of deubiquitylating enzymes as drug targets, there is increasing demand for versatile high-throughput (HT) tools for the identification of potent and selective DUB modulators. Here we present the HT adaptation of the previously published MALDI-TOF–based DUB assay method. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains by DUBs. The method has been specifically developed for use with nanoliter-dispensing robotics to meet drug discovery requirements for the screening of large and diverse compound libraries. Contrary to the most common DUB screening technologies currently available, the MALDI-TOF DUB assay combines the use of physiological substrates with the sensitivity and reliability of the mass spectrometry–based readout.

Publication metadata

Author(s): De Cesare V, Moran J, Traynor R, Knebel A, Ritorto MS, Trost M, McLauchlan H, Hastie CJ, Davies P

Publication type: Article

Publication status: Published

Journal: Nature Protocols

Year: 2020

Volume: 15

Pages: 4034–4057

Online publication date: 02/11/2020

Acceptance date: 26/08/2020

ISSN (print): 1754-2189

ISSN (electronic): 1750-2799

Publisher: Nature Research

URL: https://doi.org/10.1038/s41596-020-00405-0

DOI: 10.1038/s41596-020-00405-0

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