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.

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What is the advantage of ABPs over simple enzyme assays?

They report enzyme activity/inhibition inside a cellular environment.

Which ubiquitin warhead is used here?

Propargylamine (PA) on HA-Ub.

How many DUBs are seen without fractionation?

Around 30–40.

What depth was reached with fractionation in MCF-7?

74 DUBs quantified, close to 78 DUB transcripts.

What probe:lysate ratio and time are standard?

1:200 (w/w) for 45 min at 37 °C.

Why is FT671 incubated only 10 min with probe?

Long probe incubations displace bound inhibitor.

How is HA-Ub-PA finished from the thioester?

HA-Ub-MesNa plus 250 mM propargylamine, 20 min at room temperature.

Why clarify SH-SY5Y lysates at only 600 × g?

To retain USP30 bound to mitochondria.

What screening capability does ABPP-HT add?

Rapid concentration dependence and selectivity of multiple inhibitors at once.

What earlier warhead replacement launched Ub ABPs?

C-terminal Gly76 replaced with glycyl vinyl sulfone.

What cellular context is emphasized versus purified assays?

Unbiased potency/selectivity in a cellular matrix.