Membrane-mimetic thermal proteome profiling (MM-TPP) toward 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.