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
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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