Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes.
Bianca D M van TolBjorn R van DoodewaerdGuinevere S M Lageveen-KammeijerBas C JansenCami M P Talavera OrmeñoPaul J M HekkingAysegul SapmazRobbert Q KimAngeliki MoutsiopoulouDavid KomanderDana L E VergoossenGerbrand J van der Heden van NoortHuib OvaaPaul P GeurinkPublished in: Nature communications (2023)
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.
Keyphrases
- hiv testing
- genome wide
- mass spectrometry
- amino acid
- induced apoptosis
- structural basis
- men who have sex with men
- small molecule
- liquid chromatography
- high throughput
- transcription factor
- cell cycle arrest
- cell proliferation
- high resolution
- emergency department
- signaling pathway
- gas chromatography
- human immunodeficiency virus
- living cells
- oxidative stress
- capillary electrophoresis
- climate change
- hiv infected