Ion-Based Proteome-Integrated Solubility Alteration Assays for Systemwide Profiling of Protein–Molecule Interactions

Unbiased drug target
engagement deconvolution and mechanism of
action elucidation are major challenges in drug development. Modification-free
target engagement methods, such as thermal proteome profiling, have
gained increasing popularity in the last several years. However, these
methods have limitations, and, in any case, new orthogonal approaches
are needed. Here, we present a novel isothermal method for comprehensive
characterization of protein solubility alterations using the effect
on protein solubility of cations and anions in the Hofmeister series.
We combine the ion-based protein precipitation approach with Proteome-Integrated
Solubility Alteration (PISA) analysis and use this I-PISA assay to
delineate the targets of several anticancer drugs both in cell lysates
and intact cells. Finally, we demonstrate that I-PISA can detect solubility
changes in minute amounts of sample, opening chemical proteomics applications
to small and rare biological material.