Solvent-based Shift Assay in Living Cells

Biophysical proteomics assays allow for proteome-wide, label-free monitoring of ligand-induced changes in protein structure and stability, offering insights into protein-ligand interactions and modulation of biophysical properties of cellular proteins. They exploit the principle that compound-induced alterations in structure or stability of proteins can be detected through changes in their susceptibility to denaturation. Here we introduce SPICE (Solvent Proteome Profiling In Cells), which employs solvent-based denaturation of proteins under otherwise physiological conditions in intact cells. We characterized solvent-induced denaturation of proteins inside cells as distinct from that in cell extracts and validated SPICE by detecting known drug-target interactions for multiple compound classes. We showed that SPICE can be flexibly used in different formats, to resolve denaturation or concentration-response curves upon treatment of live cells. Our results indicate that SPICE, unlike experiments in cell extracts, also detects secondary compound-induced effects such as target profiles of drug metabolites, modulation of protein-protein interactions, and downstream signalling events.