High-Throughput MALDI-TOF Mass Spectrometry-Based Deubiquitylating Enzyme Assay for Drug Discovery

Virginia De Cesare (Lead / Corresponding author), Paul Davies (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

2 Citations (Scopus)


Ubiquitylation (or ubiquitination) is the reversible conjugation of a 76-amino-acid polypeptide (ubiquitin) to a target protein to modulate various biological processes. Deubiquitylating enzymes (DUBs) are a class of enzymes that specifically remove ubiquitin from a substrate. In recent years DUBs have garnered significant attention as a new class of targets in multiple therapeutic areas. The recent development of high-throughput Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has provided new tools to perform drug discovery screening. Here we present a facile and high-throughput step-by-step protocol of the MALDI-TOF MS-based DUB assay for screening the activity of DUBs in vitro. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains. The presented protocol takes advantage of nanoliter dispensing robotics and automated MALDI-TOF MS analysis to screen large and diverse compound libraries.

Original languageEnglish
Title of host publicationDeubiquitinases
Subtitle of host publicationMethods and Protocols
EditorsJulie Maupin-Furlow, Mariola J. Edelmann
Place of PublicationNew York
PublisherHumana Press
Number of pages12
ISBN (Electronic)9781071628034
ISBN (Print)9781071628027
Publication statusPublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
  • High-Throughput Screening Assays/methods
  • Drug Discovery/methods
  • Ubiquitin/metabolism
  • Enzyme Assays
  • Ubiquitin
  • DUBs
  • Deubiquitylating enzyme
  • High-throughput screening
  • MALDI-TOF mass spectrometry
  • Drug discovery

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology


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