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. These assays 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 solvent proteome profiling in cells (SPICE), 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. 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 signaling events. We further demonstrate complementarity of SPICE and cellular thermal shift assay, which both robustly detect the designated targets of well-characterized drugs and individually provide biologically meaningful and interpretable results. Finally, we show that SPICE can detect covalent drug-targets, compound-induced target-destabilization and stabilization of degrader drug targets despite their concurrent degradation.

D. Steinbrunn, C. Cepeleaga, A. Betz, G. Kibar, M. Holzner, S. K. Maier, C. Zasada, G. Hagemann, S. A. Sieber, and H. Hahne. "Proteome-Wide Monitoring of Drug Action in Living Cells Using a Novel Label-Free Solvent-Based Shift Assay." Molecular & Cellular Proteomics, 24(12), 101444 (2025).

Link: https://doi.org/10.1016/j.mcpro.2025.101444

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