Membrane mimetic thermal proteome profiling (MM-TPP) towards mapping membrane protein-ligand dynamic interactions

Integral membrane proteins (IMPs) are central targets for small-molecule therapeutics, yet robust, unbiased, and detergent-free approaches to assess their on- and off-target interactions remain limited. Previously, we introduced the Peptidisc membrane mimetic (MM) for water-soluble stabilization of the membrane proteome and interactome (Carlson et al., eLife, 2019). In this work, we combine the Peptidisc with thermal proteome profiling (TPP) to establish membrane mimetic thermal proteome profiling (MM-TPP), a method that enables proteome-wide mapping of membrane protein–ligand interactions. Using a membrane protein library derived from mouse liver tissue, we detected the specific effects of ATP and orthovanadate on the thermal stability of ATP-binding cassette (ABC) transporters, as well as stability shifts driven by the hydrotropic effect of ATP and its by-products on G protein–coupled receptors (GPCRs). In contrast, detergent-based TPP (DB-TPP) with ATP–VO₄ failed to yield specific enrichment of ATP-binding proteins, underscoring the unique capacity of MM-TPP. To further validate the approach, we demonstrated the ability of MM-TPP to detect specific ligand-induced stabilization of cognate targets, exemplified by the selective thermal stabilization of the P2RY12 receptor by 2-methylthio-ADP. Together, these findings position MM-TPP as a robust platform for uncovering both on- and off-target effects of small molecules, providing insights into the druggable membrane proteome and its stability in consequence of changing dynamic ligands.