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.