Abstract
We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer198*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.
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Abbreviations
- AChE:
-
Acetylcholinesterase
- ACN:
-
Acetonitrile
- BChE:
-
Butyrylcholinesterase
- CE:
-
Collision energy
- cHMP:
-
O-cyclohexyl methylphosphonyl moiety
- CVX:
-
Chinese VX
- CWA:
-
Chemical warfare agents
- CWC:
-
Chemical Weapons Convention
- d 3-Atr:
-
Triple deuterated atropine
- EDMAP:
-
O-ethyl-dimethylaminophosphonyl moiety
- EMP:
-
O-ethyl-methylphosphonyl moiety
- ESI:
-
Electrospray ionisation
- FA:
-
Formic acid
- GA:
-
Nerve agent tabun
- GB:
-
Nerve agent sarin
- GD:
-
Nerve agent soman
- GF:
-
Nerve agent cyclosarin
- HR:
-
High-resolution
- IMS:
-
Immunomagnetic separation
- iPrOH:
-
Iso-propanol
- k i :
-
Inhibition rate constant
- LC:
-
Liquid chromatography
- LOI:
-
Limit of identification
- M :
-
Mean
- MS:
-
Mass spectrometry
- MS/MS:
-
Tandem mass spectrometry
- NP:
-
Nonapeptide
- OP:
-
Organophosphorus
- OPCW:
-
Organisation for the Prohibition of Chemical Weapons
- OPNA:
-
Organophosphorus nerve agents
- PIS:
-
Product ion scan
- pm :
-
Phosphyl moiety
- Prot K:
-
Proteinase K
- Qual:
-
Qualifying ion
- RVX:
-
Russian VX
- RSD:
-
Relative standard deviation
- SD:
-
Standard deviation
- SRM:
-
Selected reaction monitoring
- TP:
-
Tetrapeptide
- UF:
-
Ultrafiltration
- VX:
-
Nerve agent O-ethyl-S-(2-diisopropylaminoethyl)methylphosphono thioate
- XIC:
-
Extracted ion chromatogram
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Part of the work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, Grant/Award Number Research Training Group GRK 2338, P03).
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Kranawetvogl, T., Siegert, M., Steinritz, D. et al. The phosphylated butyrylcholinesterase-derived tetrapeptide GlyGluSerAla proves exposure to organophosphorus agents with enantioselectivity. Arch Toxicol 98, 791–806 (2024). https://doi.org/10.1007/s00204-023-03657-3
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DOI: https://doi.org/10.1007/s00204-023-03657-3