Chemoproteomics Prioritizes Mitochondrial ADP/ATP Translocase as a Candidate Target Associated with 6PPDQ-Induced...

6PPD quinone (6PPDQ) is an emerging contaminant that induces acute respiratory toxicity in rainbow trout (Oncorhynchus mykiss), yet its underlying molecular mechanisms remain poorly understood. In the present study, short-term in vivo exposure of rainbow trout to 6PPDQ resulted in substantial accumulation and limited biotransformation of 6PPDQ in the gill, accompanied by pronounced gill structural damage and increased whole-fish oxygen consumption. Taking advantage of the electrophilic reactivity of the quinone moiety of 6PPDQ toward cysteine residues, we applied activity-based protein profiling (ABPP) to gill tissue. ABPP revealed marked alterations in mitochondrial cysteine reactivity and highlighted ADP/ATP translocase (ANT) as a candidate 6PPDQ-interacting mitochondrial protein. A Cys-160-containing ANT peptide within the nucleotide-binding domain of ANT was pinpointed as the covalent binding site through ABPP, Peptide-centric Local Stability Assay (PELSA), and molecular docking. Functional assays using isolated gill mitochondria showed that 6PPDQ elicited an uncoupling-like mitochondrial respiratory response that was partially attenuated by the ANT inhibitor carboxyatractyloside (CATR), supporting the functional involvement of ANT in this gill-based model. Together, these findings nominate ANT as a candidate gill mitochondrial target associated with 6PPDQ-induced acute respiratory toxicity and demonstrate the utility of chemoproteomics for prioritizing mechanistically relevant protein interactions of emerging pollutants.