Protein adduct formation between RuBisCO and V-type organophosphorus nerve agents

Organophosphorus nerve agents (OPNAs) are highly toxic compounds that disrupt the central nervous system through irreversible inhibition of acetylcholinesterase. Classified as chemical weapons under the Chemical Weapons Convention (CWC), OPNAs necessitate reliable and extended-window detection methods for exposure verification. To expand the range of biomarker candidates for OPNA exposure in food matrices, this study investigated the in vitro formation of adducts between ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-a predominant protein in spinach-and V-type OPNAs. Mass spectrometry identified 13 reproducibly adducted peptides; notably, 5 peptides showed conserved covalent modification sites across all three OPNAs investigated in this study (VX, VR, and Vs), indicating agent-independent targeting. The modified sequences GHYLNATAGTCEDMMK (Y239/C247), WSPELAAACEVWK (C459), YGRPLLGCTIKPK (Y165), and FLFC∗AEALYK (C221) exhibited high adduct stability. These stable and modified peptides are promising biomarkers for OPNA-exposure tracking. Thus, this study provides a basis for the forensic analysis of organophosphorus toxicant residues and food safety incident investigations.