Chemoproteomic profiling by bioorthogonal probes to reveal the novel targets of acrylamide in microglia

Neurotoxicity studies caused by exposure to acrylamide (AA) are of wide interest, but the methods for direct analysis of AA targets in living neuronal cells by cysteine profiling are still lacking. To address this, we developed a specific bioorthogonal probe, AAPA-P2, for chemical proteomics analysis of AA covalent binding sites. AAPA-P2 captured 754 target proteins, increasing the number of identified target proteins by 20-fold. Further screening revealed 96 proteins that are both highly sensitive and heavily modified by AAPA-P2, with validation performed on some potential key targets and binding sites. AA was found to induce neurotoxicity by binding to newly identified targets, Proteasome 26S Subunit, non ATPase 9 (PSMD9) and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5 (NDUFA5), interfering with the ubiquitin-proteasome system, and inducing mitochondria-dependent apoptosis. The present work provides an effective bioorthogonal probe tool for identifying covalent binding targets of acrylamide and offers new insights into the molecular mechanisms underlying acrylamide-induced neurotoxicity.