Identification of Butyrylcholinesterase-Derived Small Molecule Peptides Indicative of Novichok Nerve Agent Exposures

Novichok nerve agents, such as A-230, A-232, and A-234, were classified as Schedule 1 chemicals under the Chemical Weapons Convention (CWC) by the Organisation for the Prohibition of Chemical Weapons (OPCW) following poisoning incidents in 2018. As a result, the production, storage, and use of these chemicals are strictly prohibited by CWC signatory nations. The identification of biomarkers indicating Novichok exposure in humans is crucial for prompt detection and response to potential incidents involving these banned chemical weapons. In this study, BChE was isolated from human serum samples exposed to Novichok nerve agents in vitro using immunomagnetic capture, followed by enzymatic digestion with Pronase or proteinase K to generate new peptide biomarkers indicative of exposure. We identified nine previously unpublished Novichok-adducted peptides generated through enzymatic digestion with proteinase K and Pronase using liquid chromatography-high-resolution mass spectrometry. Two peptides, [Agent]-serine-alanine for proteinase K digestion and [Agent]-serine-alanine-glycine for Pronase digestion, were selected for optimization due to their abundance. The analysis was subsequently transferred to an LC-triple quadrupole system to enhance throughput and detect these new biomarkers at the limits of detection corresponding to BChE inhibition levels of 3.90% or less. These additional biomarkers can improve laboratory preparedness for OPCW-designated biomedical testing laboratories as well as other clinical and investigative laboratories tasked with responding to emergencies involving these highly toxic chemicals.