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

Integral membrane proteins (IMPs) are key targets for small-molecule therapeutics. However, robust, unbiased, and detergent-free methods to probe on- and off-target interactions within this protein class remain underdeveloped. Previously, we introduced the Peptidisc membrane mimetic (MM) for water-soluble stabilization of the membrane proteome and interactome (Carlson et al., 2019). Here, we integrate the Peptidisc with thermal proteome profiling (TPP) to establish membrane mimetic thermal proteome profiling (MM-TPP), a method that enables proteome-wide characterization of membrane protein–ligand interactions. Using a membrane protein library derived from the mouse liver, we assessed the specific effects of ATP and orthovanadate on the thermal stability of ATP-binding cassette (ABC) transporters, but also stability shifts driven by the hydrotropic effect of ATP and its by-products on G protein–coupled receptors (GPCRs). Conversely, when tested in detergent-based TPP (DB-TPP) using ATP–VO₄, no specific enrichment of ATP-binding protein were obtained, highlighting the utility of MM-TPP. As an additional validation step, we tested MM-TPP’s ability to capture specific ligand-induced stabilization of cognate targets, here demonstrated by the selective thermal stabilization of the P2RY12 receptor by 2-methylthio-ADP. Altogether, MM-TPP emerges as a robust platform for identifying both on- and off-target effects of small molecules, offering new insights into the druggable membrane proteome and its stability landscape as a consequence of changing and often dynamic small molecules.