Applying Thermal Proteome Profiling to Per- and Polyfluoroalkyl Substances (PFAS): Proteome-Wide Identification of Molecular Targets and Early Events

Conventional target screening repertoires provide limited coverage of proteome-level interactions, leaving critical gaps in the mechanistic toxicology of per- and polyfluoroalkyl substances (PFAS). In this study, thermal proteome profiling (TPP) was applied for unbiased, proteome-wide characterization of PFAS–protein interactions across five representative PFAS, identifying 173 proteins with significant ligand-induced stabilization. Specifically, legacy PFAS converged on small COPII coat GTPase SAR1A/SAR1B, consistent with potential mechanistic Target of Rapamycin (mTOR)-linked metabolic reprogramming, whereas the replacement ether PFAS hexafluoropropylene oxide dimer acid (HFPO–DA, commercially known as GenX) showed a distinct targetome highlighted by WD repeat-containing protein 89 (WDR89), suggesting non-nuclear-receptor mechanisms plausibly related to chromatin/complex assembly. Cellular thermal shift assay and molecular docking independently verified target engagement and provided a structural rationale for the observed stabilization patterns. Further ontology-based annotation linked the stabilized targets to 279 standardized disease entities, with a predominance of neoplastic outcomes. These findings demonstrate TPP as a new approach methodology for PFAS target discovery, reveal divergent early events for legacy versus replacement chemistries, and provide a proteome-scale framework to prioritize mechanism-based validation and to support evidence-weighted risk assessment of emerging fluorinated alternatives.