Ion-based Proteome Integrated Solubility Alteration Assay for System-wide 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 over 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 this ion-based protein precipitation approach with Proteome Integrated Solubility Alteration (PISA) analysis and use this I-PISA assay to delineate the targets of several anti-cancer drugs both in cell lysate and in living cells. Lastly, we demonstrate that I-PISA can detect solubility changes in minute amounts of sample, opening chemical proteomics applications to small and rare biological material.