The phosphylated butyrylcholinesterase-derived tetrapeptide GlyGluSerAla proves exposure to organophosphorus agents with enantioselectivity

We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer^198*Ala generated from butyrylcholinesterase (BChE) with proteinase K excellently suited for the verification of exposure to toxic organophosphorus nerve agents (OPNA). Verification requires bioanalytical methods mandatory for toxicological and legal reasons. OPNA react with BChE by phosphonylation of the active site serine residue (Ser¹⁹⁸) forming one of the major target protein adducts for verification. After its enzymatic cleavage with pepsin, the nonapeptide (NP) PheGlyGluSer^*AlaGlyAlaAlaSer is typically produced as biomarker. Usually OPNA occur as racemic mixtures of phosphonic acid derivatives with the stereocenter at the phosphorus atom, e.g. (±)-VX. Both enantiomers react with BChE, but the adducted NP does not allow their chromatographic distinction. In contrast, the herein introduced TP-adducts appeared as two peaks when using a stationary reversed phase (1.8 µm) in micro-liquid chromatography–electrospray ionisation tandem-mass spectrometry (µLC–ESI MS/MS) analysis. These two peaks represent diastereomers of the (+)- and (−)-OPNA adducted to the peptide that comprises chiral L-amino acids exclusively. Concentration- and time-dependent effects of adduct formation with (±)-VX and its pure enantiomers (+)- and (−)-VX as well as with (±)-cyclosarin (GF) were investigated in detail characterising enantioselective adduct formation, stability, ageing and spontaneous reactivation. The method was also successfully applied to samples from a real case of pesticide poisoning as well as to samples of biomedical proficiency tests provided by the Organisation for the Prohibition of Chemical Weapons.