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

Virginia De Cesare (Lead / Corresponding author), Jennifer Moran, Ryan Traynor, Axel Knebel, Maria Stella Ritorto, Matthias Trost, Hilary McLauchlan, C. James Hastie, Paul Davies (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Number of pages26
JournalNature Protocols
Early online date2 Nov 2020
DOIs
Publication statusE-pub ahead of print - 2 Nov 2020

Keywords

  • Deubiquitylating enzymes
  • Drug discovery
  • Mass spectrometry
  • Target validation

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