Chemical biology
High-throughput ABPP of DUB inhibitors in lysates
Open access · cc by · source: Europe PMC
A ubiquitin-propargylamine activity probe plus MS screens how selective DUB inhibitors are in a cellular protein matrix.
How this study connects
Role on claims
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A ubiquitin-propargylamine activity probe plus MS screens how selective DUB inhibitors are in a cellular protein matrix.
Evidence for the claim as stated.
Study at a glance
- Design
- Animal / in-vitro — HA-Ub-PA activity-based probe with immunopurification and MS of DUB adducts ± inhibitors
- N
- MCF-7 and SH-SY5Y lysates plus mouse tissue extracts; ~30–40 DUBs quantified without fractionation
- Population
- Human cell lysates and mouse tissue in a cellular protein matrix
- Outcome
- High-throughput ABPP maps concentration-dependent selectivity of DUB inhibitors, including USP30 compounds
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Inspect source passages
High-throughput ABPP of DUB inhibitors in lysates
These passages occur verbatim in the stored paper text. They support the points listed below; they do not verify every statement in the explanation.
Without fractionation about 30–40 DUBs are immunoprecipitated and quantified.
Without fractionation the number of DUBs immunoprecipitated and quantified by MS with a propargylamine (PA) warhead is around 30–40 (; ).
With fractionation, 74 DUBs in MCF-7 cells were quantified versus 78 DUB transcripts.
we have recently combined this probe (Ub-PA) with sample fractionation and 74 DUBs in MCF-7 breast cancer cells were quantified. For comparison, the transcriptomics analysis of the same cells identified a very similar number of (78) DUB
Key findings
Without fractionation ~30–40 DUBs are quantified; with fractionation they previously reached 74 DUBs in MCF-7 (similar to 78 transcripts). The HT format maps concentration dependence and selectivity of several inhibitors across cell types.
Methodology
Authors labeled MCF-7/SH-SY5Y (and mouse tissue) lysates with HA-Ub-PA, immunopurified adducts, and quantified DUBs by MS with or without inhibitors (including USP30 compounds), using short incubations so the covalent probe does not outcompete tight inhibitors.
Limitations
This is lysate ABPP, not proof of in vivo efficacy or safety; probe occupancy is a surrogate for inhibition and can displace some inhibitors if incubation is too long.
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