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Mass spectrometry based proteomic approach for the screening of butyrylcholinesterase adduct formation with organophosphates.

Talanta (2019-02-18)
Yaroslav Dubrovskii, Ekaterina Murashko, Olga Chuprina, Petr Beltyukov, Andrey Radilov, Nikolay Solovyev, Vladimir Babakov
ABSTRACT

Organophosphates' toxic effect causes covalent binding to serine-198 in the active site of human plasma butyrylcholinesterase (BChE) with loss of enzymatic function (covalent inhibition). Mass spectrometric detection of modified FGESAGAAS peptide at the active site is a powerful exposure biomarker tool. The aim of this study was to develop mass spectrometry-based method for BChE adduct formation screening, avoiding the use of standard peptides. Immunomagnetic separation of proteins from plasma was optimized. Commercially available anti-butyrylcholinesterase monoclonal antibodies, immobilized on magnetic beads, resulted in stable and reusable affinity sorbent. The method was tested on horse serum BChE and real human plasma from healthy donors, treated with Russian VX (VR). The BChE isolated from blood plasma was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method was evaluated by using synthetic peptides and by comparison to the enzymatic activity Ellman's assay. The minimum concentration of VR exposure, resulting in detectable VR-adduct, was 0.2 ng/mL, which corresponded to the relative BChE inhibition of less than 2%. Adduct formation assessment was performed via monitoring of decrease in non-modified peptide LC-MS/MS signal and increase in VR-modified peptide signal. The designed approach was tested in a pilot study with 5 blood samples from healthy volunteers. Mass spectrometry-based method for BChE adduct formation was found to be in agreement with Ellman's inhibition assay, so the method is applicable for direct BChE inhibition assessment.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ammonium bicarbonate, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Pepsin from porcine gastric mucosa, lyophilized powder, ≥3,200 units/mg protein
Sigma-Aldrich
Phosphate Buffered Saline, 10× PBS for Western blots and IP
Sigma-Aldrich
Butyrylcholinesterase from equine serum, lyophilized powder, ≥900 units/mg protein