1. Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes.
- Author
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van Tol BDM, van Doodewaerd BR, Lageveen-Kammeijer GSM, Jansen BC, Talavera Ormeño CMP, Hekking PJM, Sapmaz A, Kim RQ, Moutsiopoulou A, Komander D, Wuhrer M, van der Heden van Noort GJ, Ovaa H, and Geurink PP
- Subjects
- Ubiquitination, Ubiquitin metabolism, Ubiquitins metabolism, Polyubiquitin metabolism, Deubiquitinating Enzymes metabolism
- Abstract
Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration., (© 2023. The Author(s).)
- Published
- 2023
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