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Lookup NU author(s): Professor Matthias TrostORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2018 Elsevier Ltd Due to their role in many diseases, enzymes of the ubiquitin system have recently become interesting drug targets. Despite efforts, primary screenings of compound libraries targeting E2 enzymes and E3 ligases have been strongly limited by the lack of robust and fast high-throughput assays. Here we report a label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry. The MALDI-TOF E2/E3 assay allows testing E2 enzymes and E3 ligases for their ubiquitin transfer activity, identifying E2/E3 active pairs, inhibitor potency and specificity and screening compound libraries in vitro without chemical or fluorescent probes. We demonstrate that the MALDI-TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared with standard biochemical assays. Due to their role in many diseases, there is a need to identify drug candidates for enzymes of the ubiquitin system in robust and high-throughput assays. Here we report as label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry.
Author(s): De Cesare V, Johnson C, Barlow V, Hastie J, Knebel A, Trost M
Publication type: Article
Publication status: Published
Journal: Cell Chemical Biology
Year: 2018
Volume: 25
Issue: 9
Pages: 1117-1127.e4
Print publication date: 20/09/2018
Online publication date: 12/07/2018
Acceptance date: 08/06/2018
Date deposited: 16/07/2018
ISSN (print): 2451-9456
ISSN (electronic): 2451-9448
Publisher: Cell Press
URL: https://doi.org/10.1016/j.chembiol.2018.06.004
DOI: 10.1016/j.chembiol.2018.06.004
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