Integrated Protein Solubility Shift Assays for Comprehensive Drug Target Identification on a Proteome-Wide Scale

Target proteins are often stabilized after binding with
a ligand
and thereby typically become more resistant to denaturation. Based
on this phenomenon, several methods without the need to covalently
modify the ligand have been developed to identify target proteins
for a specific ligand. These methods usually employ complicated workflows
with high cost and limited throughput. Here, we develop an iso-pH
shift assay (ipHSA) method, a proteome-wide target identification
method that detects ligand-induced protein solubility shifts by precipitating
proteins with a single concentration of acidic agent followed by protein
quantification via data-independent acquisition (DIA). Using a pan-kinase
inhibitor, staurosporine, we demonstrated that ipHSA increased throughput
compared to the previously developed pH-dependent protein precipitation
(pHDPP) method. ipHSA was found to have high complementarity in staurosporine
target identification compared with the improved isothermal shift
assay (iTSA) and isosolvent shift assay (iSSA) using DIA instead of
tandem mass tags (TMTs) for quantification. To further improve target
identification sensitivity, we developed an integrated protein solubility
shift assay (IPSSA) by pooling the supernatants yielded from ipHSA,
iTSA, and iSSA methods. IPSSA exhibited increased sensitivity in screening
staurosporine targets by 38, 29, and 38% compared to individual methods.
Increasing the number of replicate experiments further enhanced the
sensitivity of target identification. Meanwhile, IPSSA also improved
the throughput and reduced the cost compared with previous methods.
As a fast and efficient tool for drug target identification, IPSSA
is expected to have broad applications in the study of the mechanism
of action.