Integrative Chemical Proteomics-Metabolomics Approach Reveals Acaca/Acacb as Direct Molecular Targets of PFOA

Identification of the direct molecular targets of environmental pollutants is of great importance for toxicity mechanism studies. Despite numerous studies have been conducted to investigate the toxicity mechanism of perfluorinated compounds (PFCs), their direct-binding protein targets which trigger downstream toxicity effects remain largely unknown. Herein, we present a systematic chemical proteomic study to profile the target proteins of PFCs by taking PFOA as a representative. Considering its electrophilicity, PFOA could preferentially bind to reactive cysteine-containing proteins. Therefore, two complementary cysteine-targeting probes, iodoacetamide alkyne (IAA) and ethynyl benziodoxolone azide (EBX), were selected to enrich the putative target proteins in the absence or presence of PFOA. Quantitative proteomic analysis of the enriched proteins identified Acaca and Acacb as novel target proteins of PFOA. We then applied parallel reaction monitoring (PRM)-based targeted proteomics study combined with thermal shift assay-based chemical proteomics to verify Acaca and Acacb as bona fide binding targets. These findings afford a plausible explanation for the PFOA-induced liver toxicity, especially regarding abnormal fatty acid metabolism that was validated by targeted metabolomics analysis. The present study documents an integrative chemical proteomics-metabolomics platform that facilitates the authentic identification of proteins that are 'targeted by small molecules and its potential to be applied for toxicity mechanism studies of environmental pollutants.