High-throughput ABPP of DUB inhibitors in lysates
A ubiquitin-propargylamine activity probe plus MS screens how selective DUB inhibitors are in a cellular protein matrix.
Source
ABPP-HT - High-Throughput Activity-Based Profiling of Deubiquitylating Enzyme Inhibitors in a Cellular Context
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
What they did
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.
What they found
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.
The limits
What it doesn't show
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.
Key terms
- Activity-based probe (ABP)
- Covalent warhead plus tag that labels active enzymes in a proteome.
- DUB
- Deubiquitylating enzyme that removes ubiquitin from substrates.
- HA-Ub-PA
- HA-tagged ubiquitin with a C-terminal propargylamine warhead for DUB active-site cysteines.
- ABPP
- Activity-based protein profiling: probe labeling, enrichment, and MS.
- USP30
- Mitochondria-associated DUB targeted by inhibitors in this screen.
Flashcards
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Quiz yourself
HA-Ub-PA labels DUBs via a:
Common questions
What warhead is on the ubiquitin probe?
Propargylamine (PA) at the C-terminus.
How many DUBs without fractionation?
Around 30–40.
Why shorten probe time for FT671?
Long incubations displace that bound inhibitor.
Why SH-SY5Y low-speed spin?
To retain mitochondrial USP30.
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