Analytical and Bioanalytical Chemistry

, Volume 406, Issue 21, pp 5171–5185 | Cite as

V-type nerve agents phosphonylate ubiquitin at biologically relevant lysine residues and induce intramolecular cyclization by an isopeptide bond

  • Christian Schmidt
  • Felicitas Breyer
  • Marc-Michael Blum
  • Horst Thiermann
  • Franz Worek
  • Harald John
Research Paper
Part of the following topical collections:
  1. Analysis of Chemicals Relevant to the Chemical Weapons Convention


Toxic organophosphorus compounds (e.g., pesticides and nerve agents) are known to react with nucleophilic side chains of different amino acids (phosphylation), thus forming adducts with endogenous proteins. Most often binding to serine, tyrosine, or threonine residues is described as being of relevance for toxicological effects (e.g., acetylcholinesterase and neuropathy target esterase) or as biomarkers for post-exposure analysis (verification, e.g., albumin and butyrylcholinesterase). Accordingly, identification of novel protein targets might be beneficial for a better understanding of the toxicology of these compounds, revealing new bioanalytical verification tools, and improving knowledge on chemical reactivity. In the present study, we investigated the reaction of ubiquitin (Ub) with the V-type nerve agents Chinese VX, Russian VX, and VX in vitro. Ub is a ubiquitous protein with a mass of 8564.8 Da present in the extra- and intracellular space that plays an important physiological role in several essential processes (e.g., proteasomal degradation, DNA repair, protein turnover, and endocytosis). Reaction products were analyzed by matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF MS) and μ-high-performance liquid chromatography online coupled to UV-detection and electrospray ionization MS (μHPLC-UV/ESI MS). Our results originally document that a complex mixture of at least mono-, di, and triphosphonylated Ub adducts was produced. Surprisingly, peptide mass fingerprint analysis in combination with MALDI and ESI MS/MS revealed that phosphonylation occurred with high selectivity in at least 6 of 7 surface-exposed lysine residues that are essential for the biological function of Ub. These reaction products were found not to age. In addition, we herein report for the first time that phosphonylation induced intramolecular cyclization by formation of an isopeptide bond between the ε-amino group of a formerly phosphonylated lysine and the side chain of an adjacent acidic glutamic acid residue.

Lysine residues in ubiquitin are phosphonylated by nerve agents and undergo intramolecular cyclization


Ubiquitin Phosphonylation Protein adducts MALDI MS LC-ESI MS Peptide mass fingerprint 









Chinese VX


Electrospray ionization


Formic acid


Endoproteinase Glu-C


High-performance liquid chromatography


Liquid chromatography


Matrix-assisted laser desorption/ionization


Molecular dynamics


Mass spectrometry


Tandem mass spectrometry


Molecular weight


Molecular weight cut-off


Neuropathy target esterase


Organophosphorus compounds


Peptide mass fingerprint


Russian VX


Total ion chromatogram


Trifluoroacetic acid








Ultra violet


Extracted ion chromatogram



This study was carried out as part of the work toward the doctoral thesis of Christian Schmidt.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Christian Schmidt
    • 1
  • Felicitas Breyer
    • 2
  • Marc-Michael Blum
    • 3
  • Horst Thiermann
    • 1
  • Franz Worek
    • 1
  • Harald John
    • 1
  1. 1.Bundeswehr Institute of Pharmacology and ToxicologyMunichGermany
  2. 2.Faculty of Natural SciencesUniversity of UlmUlmGermany
  3. 3.Organisation for the Prohibition of Chemical Weapons (OPCW), OPCW LaboratoryRijswijkThe Netherlands

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