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Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma

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Abstract

Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS2 analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS2 analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL−1). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction.

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase

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References

  1. Wang J, Timchalk C, Lin Y. Carbon nanotube-based electrochemical sensor for assay of salivary cholinesterase enzyme activity: an exposure biomarker of organophosphate pesticides and nerve agents. Environ Sci Technol. 2008;42(7):2688–93.

    Article  CAS  Google Scholar 

  2. Raveh L, Grunwald J, Marcus D, Papier Y, Cohen E, Ashani Y. Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity. In vitro and in vivo quantitative characterization. Biochem Pharmacol. 1993;45(12):2465–74.

    Article  CAS  Google Scholar 

  3. Williams NH, Harrison JM, Read RW, Black RM. Phosphylated tyrosine in albumin as a biomarker of exposure to organophosphorus nerve agents. Arch Toxicol. 2007;81(9):627–39.

    Article  CAS  Google Scholar 

  4. Marsillach J, Hsieh EJ, Richter RJ, MacCoss MJ, Furlong CE. Proteomic analysis of adducted butyrylcholinesterase for biomonitoring organophosphorus exposures. Chem Biol Interact. 2013;203(1):85–90.

    Article  CAS  Google Scholar 

  5. Carletti E, Aurbek N, Gillon E, Loiodice M, Nicolet Y, Fontecilla-Camps J-C, et al. Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun. Biochem J. 2009;421(1):97–106.

    Article  CAS  Google Scholar 

  6. Masson P, Carletti E, Nachon F. Structure, activities and biomedical applications of human butyrylcholinesterase. Protein Pept Lett. 2009;16(10):1215–24.

    Article  CAS  Google Scholar 

  7. Sporty JLS, Lemire SW, Jakubowski EM, Renner JA, Evans RA, Williams RF, et al. Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum. Anal Chem. 2010;82(15):6593–600.

    Article  CAS  Google Scholar 

  8. Fidder A, Hulst AG, Noort D, de Ruiter R, van der Schans MJ, Benschop HP, et al. Retrospective detection of exposure to organophosphorus anti-cholinesterases: mass spectrometric analysis of phosphylated human butyrylcholinesterase. Chem Res Toxicol. 2002;15(4):582–90.

    Article  CAS  Google Scholar 

  9. Black RM. History and perspectives of bioanalytical methods for chemical warfare agent detection. J Chromatogr B. 2010;878(17–18):1207–15.

    Article  CAS  Google Scholar 

  10. Black RM, Clarke RJ, Read RW, Reid MTJ. Application of gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry to the analysis of chemical warfare samples, found to contain residues of the nerve agent sarin, sulphur mustard and their degradation products. J Chromatogr A. 1994;662(2):301–21.

    Article  CAS  Google Scholar 

  11. Fredriksson S-Å, Hammarström L-G, Henriksson L, Lakso H-Å. Trace determination of alkyl methylphosphonic acids in environmental and biological samples using gas chromatography/negative-ion chemical ionization mass spectrometry and tandem mass spectrometry. J Mass Spectrom. 1995;30(8):1133–43.

    Article  CAS  Google Scholar 

  12. Black RM, Read RW. Application of liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry, and tandem mass spectrometry, to the analysis and identification of degradation products of chemical warfare agents. J Chromatogr A. 1997;759(1–2):79–92.

    Article  CAS  Google Scholar 

  13. Tørnes JA. Identification of some alkyl methylphosphonic acids by thermospray tandem mass spectrometry. Rapid Commun Mass Spectrom. 1996;10(8):878–82.

    Article  Google Scholar 

  14. Noort D, Hulst AG, Platenburg DH, Polhuijs M, Benschop HP. Quantitative analysis of O-isopropyl methylphosphonic acid in serum samples of Japanese citizens allegedly exposed to sarin: estimation of internal dosage. Arch Toxicol. 1998;72(10):671–5.

    Article  CAS  Google Scholar 

  15. Black RM, Read RW. Analysis of degradation products of organophosphorus chemical warfare agents and related compounds by liquid chromatography–mass spectrometry using electrospray and atmospheric pressure chemical ionisation. J Chromatogr A. 1998;794(1–2):233–44.

    Article  CAS  Google Scholar 

  16. Swaim LL, Johnson RC, Zhou Y, Sandlin C, Barr JR. Quantification of organophosphorus nerve agent metabolites using a reduced-volume, high-throughput sample processing format and liquid chromatography-tandem mass spectrometry. J Anal Toxicol. 2008;32(9):774–7.

    Article  CAS  Google Scholar 

  17. Adams TK, Capacio BR, Smith JR, Whalley CE, Korte WD. The application of the fluoride reactivation process to the detection of sarin and soman nerve agent exposures in biological samples. Drug Chem Toxicol. 2004;27(1):77–91.

    Article  CAS  Google Scholar 

  18. Pantazides BG, Watson CM, Carter MD, Crow BS, Perez JW, Blake TA, et al. An enhanced butyrylcholinesterase method to measure organophosphorus nerve agent exposure in humans. Anal Bioanal Chem. 2014;406(21):5187–94.

    Article  CAS  Google Scholar 

  19. Read RW, Riches JR, Stevens JA, Stubbs SJ, Black RM. Biomarkers of organophosphorus nerve agent exposure: comparison of phosphylated butyrylcholinesterase and phosphylated albumin after oxime therapy. Arch Toxicol. 2010;84(1):25–36.

    Article  CAS  Google Scholar 

  20. Wang L, Du D, Lu D, Lin C-T, Smith JN, Timchalk C, et al. Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: a biomarker of exposure to organophosphate agents. Anal Chim Acta. 2011;693(1–2):1–6.

    CAS  Google Scholar 

  21. Ashani Y. Prospective of human butyrylcholinesterase as a detoxifying antidote and potential regulator of controlled-release drugs. Drug Dev Res. 2000;50(3–4):298–308.

    Article  CAS  Google Scholar 

  22. Saxena A, Luo C, Doctor BP. Developing procedures for the large-scale purification of human serum butyrylcholinesterase. Protein Expr Purif. 2008;61(2):191–6.

    Article  CAS  Google Scholar 

  23. Carter MD, Crow BS, Pantazides BG, Watson CM, Thomas JD, Blake TA, et al. Direct quantitation of methyl phosphonate adducts to human serum butyrylcholinesterase by immunomagnetic-UHPLC-MS/MS. Anal Chem. 2013;85(22):11106–11.

    Article  CAS  Google Scholar 

  24. Knaack JS, Zhou Y, Abney CW, Prezioso SM, Magnuson M, Evans R, et al. High-throughput immunomagnetic scavenging technique for quantitative analysis of live VX nerve agent in water, hamburger, and soil matrixes. Anal Chem. 2012;84(22):10052–7.

    Article  CAS  Google Scholar 

  25. John H, Breyer F, Schmidt C, Mizaikoff B, Worek F, Thiermann H. Small-scale purification of butyrylcholinesterase from human plasma and implementation of a μLC-UV/ESI MS/MS method to detect its organophosphorus adducts: butyrylcholinesterase for OP adducts. Drug Test Anal. 2015;7(10):947–956.

  26. van der Schans MJ, Fidder A, van Oeveren D, Hulst AG, Noort D. Verification of exposure to cholinesterase inhibitors: generic detection of OPCW schedule 1 nerve agent adducts to human butyrylcholinesterase. J Anal Toxicol. 2008;32(1):125–30.

    Article  Google Scholar 

  27. Liu C-C, Huang G-L, Xi H-L, Liu S-L, Liu J-Q, Yu H-L, et al. Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC–MS/MS. J Chromatogr B. 2016;1036–1037:57–65.

    Article  Google Scholar 

  28. Li H, Tong L, Schopfer LM, Masson P, Lockridge O. Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase. Chem Biol Interact. 2008;175(1–3):68–72.

    Article  CAS  Google Scholar 

  29. Tsuge K, Seto Y. Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography–mass spectrometric analysis after in gel chymotryptic digestion. J Chromatogr B. 2006;838(1):21–30.

    Article  CAS  Google Scholar 

  30. Mehrani H. Simplified procedures for purification and stabilization of human plasma butyrylcholinesterase. Process Biochem. 2004;39(7):877–82.

    Article  CAS  Google Scholar 

  31. Lockridge O, Schopfer LM, Winger G, Woods JH. Large scale purification of butyrylcholinesterase from human plasma suitable for injection into monkeys: a potential new therapeutic for protection against cocaine and nerve agent toxicity. J Med Chem Biol Radiol Def. 2005;3:nihms5095.

    Google Scholar 

  32. Masson P, Sussmilch A, Charlet JP. Purification of butyrylcholinesterase from human plasma. C R Seances Acad Sci D. 1980;290(13):857–903.

    CAS  Google Scholar 

  33. Liyasova M, Li B, Schopfer LM, Nachon F, Masson P, Furlong CE, et al. Exposure to tri-o-cresyl phosphate detected in jet airplane passengers. Toxicol Appl Pharmacol. 2011;256(3):337–47.

    Article  CAS  Google Scholar 

  34. Noort D, Fidder A, van der Schans MJ, Hulst AG. Verification of exposure to organophosphates: generic mass spectrometric method for detection of human butyrylcholinesterase adducts. Anal Chem. 2006;78(18):6640–4.

    Article  CAS  Google Scholar 

  35. Brazzolotto X, Wandhammer M, Ronco C, Trovaslet M, Jean L, Lockridge O, et al. Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure: recombinant human butyrylcholinesterase in insect cells. FEBS J. 2012;279(16):2905–16.

    Article  CAS  Google Scholar 

  36. Aryal UK, Lin C-T, Kim J-S, Heibeck TH, Wang J, Qian W-J, et al. Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry. Anal Chim Acta. 2012;723:68–75.

    Article  CAS  Google Scholar 

  37. Marsillach J, Richter RJ, Kim JH, Stevens RC, MacCoss MJ, Tomazela D, et al. Biomarkers of organophosphorus (OP) exposures in humans. Neurotoxicology. 2011;32(5):656–60.

    Article  CAS  Google Scholar 

  38. Mathews TP, Carter MD, Johnson D, Isenberg SL, Graham LA, Thomas JD, et al. High-confidence qualitative identification of organophosphorus nerve agent adducts to human butyrylcholinesterase. Anal Chem. 2017;89(3):1955–64.

    Article  CAS  Google Scholar 

  39. Peng H, Brimijoin S, Hrabovska A, Targosova K, Krejci E, Blake TA, et al. Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences. Chem Biol Interact. 2015;240:336–45.

    Article  CAS  Google Scholar 

  40. Graham LA, Johnson D, Carter MD, Stout EG, Erol HA, Isenberg SL, et al. A high-throughput UHPLC-MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents: aged butyrylcholinesterase biomarkers of organophosphorus nerve agents. Biomed Chromatogr [Internet]. 2016 [cited 2017 Feb 6]. Available from: http://doi.wiley.com/10.1002/bmc.3830.

  41. Bonichon M, Combès A, Desoubries C, Bossée A, Pichon V. Development of immobilized-pepsin microreactors coupled to nano liquid chromatography and tandem mass spectrometry for the quantitative analysis of human butyrylcholinesterase. J Chromatogr A. 2016;1461:84–91.

    Article  CAS  Google Scholar 

  42. Cingöz A, Hugon-Chapuis F, Pichon V. Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry. J Chromatogr B. 2010;878(2):213–21.

    Article  Google Scholar 

  43. Peng H, Brimijoin S, Hrabovska A, Krejci E, Blake TA, Johnson RC, et al. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma. Chem Biol Interact. [Internet]. 2015 Nov [cited 2015 Dec 7]. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0009279715301137.

  44. Peng H, Blake TA, Johnson RC, Dafferner AJ, Brimijoin S, Lockridge O. Monoclonal antibodies that recognize various folding states of pure human butyrylcholinesterase can immunopurify butyrylcholinesterase from human plasma stored at elevated temperatures. ACS Omega. 2016;1(6):1182–91.

    Article  CAS  Google Scholar 

  45. Schopfer LM, Masson P, Lamourette P, Simon S, Lockridge O. Detection of cresyl phosphate-modified butyrylcholinesterase in human plasma for chemical exposure associated with aerotoxic syndrome. Anal Biochem. 2014;461:17–26.

    Article  CAS  Google Scholar 

  46. Schopfer LM, Lockridge O, Brimijoin S. Pure human butyrylcholinesterase hydrolyzes octanoyl ghrelin to desacyl ghrelin. Gen Comp Endocrinol. 2015;224:61–8.

    Article  CAS  Google Scholar 

  47. Marsillach J, Costa LG, Furlong CE. Protein adducts as biomarkers of exposure to organophosphorus compounds. Toxicology. 2013;307:46–54.

    Article  CAS  Google Scholar 

  48. John H, van der Schans MJ, Koller M, Spruit HET, Worek F, Thiermann H, et al. Fatal sarin poisoning in Syria 2013: forensic verification within an international laboratory network. Forensic Toxicol [Internet]. 2017 21 [cited 2017 Aug 3]. Available from: http://link.springer.com/10.1007/s11419-017-0376-7.

  49. Ronco C, Foucault R, Gillon E, Bohn P, Nachon F, Jean L, et al. New huprine derivatives functionalized at position 9 as highly potent acetylcholinesterase inhibitors. ChemMedChem. 2011;6(5):876–88.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Pr. Oksana Lockridge for providing mAb2 and B2 18-5 antibodies and Dr. Florian Nachon for providing huprine.

Funding

This research was supported by Direction Générale de l’Armement (DGA), grant no. 140221.

Author information

Authors and Affiliations

  1. Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM), UMR CNRS-ESPCI Paris, CBI 8231, PSL Research University, ESPCI Paris, 10 rue Vauquelin, Paris, France

    Maud Bonichon, Valentina Valbi, Audrey Combès & Valérie Pichon

  2. DGA, CBRN Defence, 5 rue Lavoisier, Vert-le-Petit, France

    Charlotte Desoubries & Anne Bossée

  3. UPMC, Sorbonne University, 4 Place Jussieu, Paris, France

    Valérie Pichon

Authors
  1. Maud Bonichon
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  2. Valentina Valbi
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  3. Audrey Combès
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  4. Charlotte Desoubries
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  5. Anne Bossée
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  6. Valérie Pichon
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Corresponding author

Correspondence to Valérie Pichon.

Ethics declarations

This is not a clinical study on humans with an ethics committee. Voluntary healthy donors (who signed a questionnaire authorizing the use of their blood bag for research purposes) donate blood to the Army Blood Transfusion Center (CTSA). DGA Maîtrise NRBC has a “Memorandum of Understanding for the transfer of products from blood or its nontherapeutic components” with the CTSA for the supply of healthy donor blood bags (number 2013-063575, March 2013). The CTSA, as a sampling agency authorized to do so, ensures donor-product-laboratory traceability and donor consent (compliance with articles R1221-22 to R1221-48 of the French public health code). Following receipt, Maîtrise NRBC polluted the blood samples in vitro that were subjected to the treatment and the analysis shown in this publication. As DGA MN is not allowed to transfer the delivered blood samples to another institution, the experiments were carried out at the DGA.

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The authors declare that they have no conflict of interest.

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Published in the topical collection celebrating ABCs 16th Anniversary.

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Bonichon, M., Valbi, V., Combès, A. et al. Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma. Anal Bioanal Chem 410, 1039–1051 (2018). https://doi.org/10.1007/s00216-017-0640-z

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  • DOI: https://doi.org/10.1007/s00216-017-0640-z

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