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Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin

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Abstract

Nerve agents still represent a serious threat to civilian and military personnel as demonstrated by the violent conflict in the Middle East. For verification of poisoning, covalent adducts with endogenous proteins (e.g., human serum albumin, HSA) are valuable long-term biomarkers. Accordingly, we developed a microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (μLC-ESI MS/HR MS) method for simultaneous detection of HSA-adducts with the V-type nerve agents VX, Chinese VX (CVX), and Russian VX (RVX). Following Pronase-catalyzed proteolysis, novel disulfide-adducts were detected in addition to phosphonylated tyrosine residues. Dipeptide disulfide-adducts were formed between the thiol-containing leaving group of the V-type nerve agents (2-(diisopropylamino)ethanethiol, DPAET, for VX and 2-(diethylamino)ethanethiol, DEAET, for CVX and RVX) and the free thiol group of Cys34 in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys448 (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys514 (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by μLC-ESI MS/HR MS. Lower limits of detection were determined in human plasma, being nearly identical for the three V-type nerve agents, and corresponded to 1–6 μM nerve agent for tyrosine-adducts, 1–3 μM nerve agent for CysPro-adducts, and 6 μM nerve agent for MetProCys-adducts, thus covering concentrations of toxicological relevance. Characterization of proteolysis kinetics revealed stable plateaus for all adducts being reached between 60 and 90 min at 37 °C. Adduct formation kinetics were characterized by simultaneously monitoring the V-type nerve agent, its leaving group, and the corresponding disulfide dimer. Furthermore, adduct formation patterns were investigated as a function of the molar ratio of HSA to V-type nerve agent.

Modification of human serum albumin (HSA) by V-type nerve agents Chinese VX (CVX) and RussianVX (RVX). Various tyrosine residues (Tyr???)n (e.g. most reactive Tyr411) were phosphonylated and disulfide-adducts were formed between the thiol-containing leaving group 2-(diethylamino)ethanethiol (DEAET) and at least three cysteine residues (Cys34, Cys448 and Cys514). Pronase-mediated proteolysis produced low-molecular cleavage products including phosphonylated tyrosines, dipeptide (Cys34Pro) and tripeptide (MetProCys448, AspIleCys514) disulfide-adducts that were detected by microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (μLC-ESI MS/HR MS).

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Abbreviations

μLC:

Microbore liquid chromatography

ACN:

Acetonitrile

APCI:

Atmospheric pressure chemical ionization

CAV:

Cell accelerator voltage

CE:

Collision energy

CP:

CysPro

CVX:

Chinese VX

CWA:

Chemical warfare agent

DEAET:

2-(Diethylamino)ethanethiol

DIC:

AspIleCys

DPAET:

2-(Diisopropylamino)ethanethiol

ESI:

Electrospray ionization

FA:

Formic acid

FV:

Fragmentor voltage

HR:

High-resolution

HSA:

Human serum albumin

LC:

Liquid chromatography

LOD:

Limit of detection

MPC:

MetProCys

MS/MS:

Tandem-mass spectrometry

OPCW:

Organisation for the Prohibition of Chemical Weapons

OPNA:

Organophosphorus nerve agent

RVX:

Russian VX

TFA:

Trifluoroacetic acid

TiPC:

Tetraisopropylcystamine

TOF:

Time-of-flight

UF:

Ultrafiltration

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Acknowledgements

JK and MG acknowledge financial support by the Deutsche Forschungsgemeinschaft under grant number FOR2372.

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Authors and Affiliations

  1. Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany

    Andreas Kranawetvogl, Franz Worek, Horst Thiermann & Harald John

  2. Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany

    Jim Küppers, Michael Gütschow & Paul W. Elsinghorst

  3. Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489, Berlin, Germany

    Markus Siegert

  4. Central Institute of the Bundeswehr Medical Service Munich, Ingolstädter Landstrasse 102, 85748, Garching, Germany

    Paul W. Elsinghorst

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  1. Andreas Kranawetvogl
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Correspondence to Harald John.

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Kranawetvogl, A., Küppers, J., Siegert, M. et al. Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin. Anal Bioanal Chem 410, 1463–1474 (2018). https://doi.org/10.1007/s00216-017-0787-7

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