High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase

Abstract

In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents.

Acetylcholinesterase: The “Hub” for Neurodegenerative Diseases and Chemical Weapons Convention

“…This reaction between BChE and neurotoxic CWC Schedule 1A not only highlights the importance of this enzyme as a biomarker, but also illustrates its potential use as a bioscavenger. Therefore, it can be a potential prophylactic measure by reacting in stoichiometric manner with nerve agents [168][169][170][171][172][173][174][175][176][177][178][179][180][181]. The knowledge on the reaction between nerve agents and cholinesterases may be useful to identify exposure to such toxic chemicals.…”

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma

“…The aged sarin and somannonapeptide adduct (m/z = 874.3) were detected with the transitions 874.3602.3 and 874.3673.3. These transitions were chosen according to previous studies on these adducts [23,25,38,40].…”

“…The aim of this study was to compare the potential of three anti-HuBuChE monoclonal antibodies (3E8, mAb2 and B2 18-5) grafted on sepharose, to extract HuBuChE and OPNAadducted HuBuChE from plasma. Antibodies 3E8 are commercially available, and were mainly used for the extraction of HuBuChE from plasma, using the immunomagnetic approach [7,18,23,24,[38][39][40]43]. The use of mAb2 and B2 18-5 antibodies for HuBuChE purification is more recent and was developed by Oksana Lockridge, using different immobilization supports like Pansorbin cells [43], Dynabeads-Protein G [39,43,44] or CNBr-activated sepharose [45,46].…”

“…Nevertheless, the antibodies fixed on the magnetic beads are digested in most of the protocols, which excludes the reusability of the beads and increases the cost of the procedure. Besides, apart from the recent work of Mathews et al [38], the immunomagnetic methods were not applied to the simultaneous detection of OPNAs and OPNA aged adducts.…”

Identification of S419 on human serum albumin as a novel biomarker for sarin and cyclosarin exposure

“…Analyses of biomedical samples, taken upon exposure, from people and animals were applied during the investigation of the use of chemical weapons . Retrospectivity was achieved by the detection of the biomarkers of nerve agent exposure .…”