High-throughput protein target mapping enables accelerated bioactivity discovery for ToxCast and PFAS compounds

Abstract

Chemical pollution is a global threat to human health, yet the toxicity mechanisms of most contaminants remains unknown. Here, we applied an ultrahigh-throughput affinity selection–mass spectrometry (AS–MS) platform to systematically identify protein targets of prioritized chemical contaminants. After benchmarking the platform, we screened 50 human proteins against 481 prioritized chemicals, including 446 ToxCast chemicals and 35 per- and polyfluoroalkyl substances (PFAS). Among 24,050 interactions assessed, we discovered 35 interactions involving 13 proteins, with fatty acid–binding proteins (FABPs) emerging as the most ligandable protein family. Given this, we selected FABPs for further validation, which revealed a distinct PFAS binding pattern: legacy PFAS selectively bound to FABP1, whereas replacement compounds, perfluoroether carboxylic acids, unexpectedly interacted with all FABPs. X-ray crystallography further revealed that the ether group enhances the molecular flexibility of alternative PFAS to accommodate the binding pockets of FABPs. Our findings demonstrate that AS–MS is a robust platform for the discovery of protein targets beyond the scope of ToxCast and highlight the broader protein-binding spectrum of alternative PFAS as potential regrettable substitutes.