Quantitative Chemical Proteomics Reveals Interspecies Variations on Binding Schemes of L-FABP with Perfluorooctanesulfonate.

Evaluating interspecies toxicity variation is a long-standing challenge for chemical hazard assessment. This study developed a quantitative interspecies thermal shift assay (QITSA) for in situ, quantitative, and modest-throughput investigation of chemical-protein interactions in cell and tissue samples across species. By using liver fatty acid binding protein (L-FABP) as a case study, the QITSA method was benchmarked with six per- and polyfluoroalkyl substances, and thermal shifts (ΔTm) were inversely related to their dissociation constants (R2 = 0.98). The QITSA can also distinguish binding modes of chemicals exemplified by palmitic acid. The QITSA was applied to determine the interactions between perfluorooctanesulfonate (PFOS) and L-FABP in liver cells or tissues from humans, mice, rats, and zebrafish. The largest thermal stability enhancement by PFOS was observed for human L-FABP followed by the mouse, rat, and zebrafish. While endogenous ligands were revealed to partially contribute to the large interspecies variation, recombinant proteins were employed to confirm the high binding affinity of PFOS to human L-FABP, compared to the rat and mouse. This study implemented an experimental strategy to characterize chemical-protein interactions across species, and future application of QITSA to other chemical contaminants is of great interest.

Nontarget Screening of Per- and Polyfluoroalkyl Substances Binding to Human Liver Fatty Acid Binding Protein.

More than 1000 per- and polyfluoroalkyl substances (PFASs) have been discovered by nontarget analysis (NTA), but their prioritization for health concerns is challenging. We developed a method by incorporating size-exclusion column co-elution (SECC) and NTA, to screen PFASs binding to human liver fatty acid binding protein (hL-FABP). Of 74 PFASs assessed, 20 were identified as hL-FABP ligands in which eight of them have high binding affinities. Increased PFAS binding affinities correlate with stronger responses in electrospray ionization (ESI-) and longer retention times on a C18 column. This is well explained by a mechanistic model, which revealed that both polar and hydrophobic interactions are crucial for binding affinities. Encouraged by this, we then developed an SECC method to identify hL-FABP ligands, and all eight high-affinity ligands were selectively captured from 74 PFASs. The method was further applied to an aqueous film-forming foam (AFFF) product in which 31 new hL-FABP ligands were identified. Suspect and nontargeted screening revealed these ligands as analogues of perfluorosulfonic acids and homologues of alkyl ether sulfates (C8- and C10/EOn, C8H17(C2H4O)nSO4-, and C10H21(C2H4O)nSO4-). The SECC method was then applied to AFFF-contaminated surface waters. In addition to perfluorooctanesulfonic acid and perfluorohexanesulfonic acid, eight other AFFF chemicals were discovered as novel ligands, including four C14- and C15/EOn. This study implemented a high-throughput method to prioritize PFASs and revealed the existence of many previously unknown hL-FABP ligands.