Skip to main content
SpringerLink
Log in

Characterization of botulinum neurotoxin type A subtypes by immunocapture enrichment and liquid chromatography–tandem mass spectrometry

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Botulinum neurotoxins (BoNT) are divided into seven toxinotypes based on their immunological properties and each toxinotype contains several subtypes according to their amino acid sequences. Here, we designed a mass spectrometry method able to identify BoNT/A subtypes in complex matrices including crude culture supernatants, food, and environmental samples. Peptides from BoNT light chain (L) specific to the subtypes BoNT/A1 to A3 and BoNT/A5 to A8 were identified. The method consists of an immunocapture step with antibodies specific to BoNT/A L chains followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) on a triple quadrupole mass spectrometer (QqQ) in multiple reaction monitoring (MRM) mode. BoNT/A subtypes were correctly identified in culture supernatants and in tap water or orange juice samples with a limit of detection of 20 to 150 mouse lethal doses (MLD) and with a lower sensitivity in serum samples.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Rossetto O, Pirazzini M, Montecucco C (2014) Botulinum neurotoxins: genetic, structural and mechanistic insights. Nat Rev Microbiol 12(8):535–549. doi:10.1038/nrmicro3295

    Article  CAS  Google Scholar 

  2. Simpson LL (2004) Identification of the major steps in botulinum toxin action. Annu Rev Pharmacol Toxicol 44:167–193

    Article  CAS  Google Scholar 

  3. Sobel J (2005) Botulism. Clin Infect Dis 41(8):1167–1173

    Article  CAS  Google Scholar 

  4. Popoff MR, Mazuet C, Poulain B (2013) Botulism and tetanus. In: The prokaryotes: human microbiology, vol 5. Human microbiology, 4° edn. Springer-Verlag, Berlin, pp 247–290

  5. Lindström M, Korkeala H (2006) Laboratory diagnosis of botulism. Clin Microbiol Rev 19(2):298–314

    Article  Google Scholar 

  6. Hill KK, Smith TJ (2013) Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes. Curr Top Microbiol Immunol 364:1–20

    Google Scholar 

  7. Smith TJ, Hill KK, Raphael BH (2014) Historical and current perspectives on Clostridium botulinum diversity. Res Microbiol 10(14):00187–00189

    Google Scholar 

  8. Smith TJ, Hill KK, Xie G, Foley BT, Williamson CH, Foster JT, Johnson SL, Chertkov O, Teshima H, Gibbons HS, Johnsky LA, Karavis MA, Smith LA (2015) Genomic sequences of six botulinum neurotoxin-producing strains representing three clostridial species illustrate the mobility and diversity of botulinum neurotoxin genes. Infect Genet Evol 30:102–113. doi:10.1016/j.meegid.2014.1012.1002

    Article  CAS  Google Scholar 

  9. Mazuet C, King LA, Bouvet P, Legeay C, Sautereau J, Popoff MR (2014) Le botulisme humain en France, 2010–2012. BEH 6:106–114

    Google Scholar 

  10. Mazuet C, Ezan E, Volland H, Popoff MR, Becher F (2012) Toxin detection in patients' sera by mass spectrometry during two outbreaks of type A botulism in France. J Clin Microbiol 50(12):4091–4094. doi:10.1128/JCM.02392-02312

    Article  Google Scholar 

  11. Kull S, Schulz KM, Weisemann J, Kirchner S, Schreiber T, Bollenbach A, Dabrowski PW, Nitsche A, Kalb SR, Dorner MB, Barr JR, Rummel A, Dorner BG (2015) Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype. PLoS One 10(2):e0116381. doi:10.1371/journal.pone.0116381

    Article  Google Scholar 

  12. Popoff MR, Bouvet P (2013) Genetic characteristics of toxigenic Clostridia and toxin gene evolution. Toxicon 75:63–89

    Article  CAS  Google Scholar 

  13. Kozaki S, Nakaue S, Kamata Y (1995) Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan. Microbiol Immunol 39(10):767–774

    Article  CAS  Google Scholar 

  14. Arndt ER, Jacobson MJ, Abola EE, Forsyth CM, Tepp WH, Marks JD, Johnson EA, Stevens ES (2006) A structural perspective of the sequence variability within botulinum neurotoxin subtypes A1–A4. J Mol Biol 362:733–742

    Article  CAS  Google Scholar 

  15. Smith TJ, Lou J, Geren IN, Forsyth CM, Tsai R, Laporte SL, Tepp WH, Bradshaw M, Johnson EA, Smith LA, Marks JD (2005) Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization. Infect Immun 73(9):5450–5457

    Article  CAS  Google Scholar 

  16. Henkel JS, Jacobson M, Tepp W, Pier C, Johnson EA, Barbieri JT (2009) Catalytic properties of botulinum neurotoxin subtypes A3 and A4. Biochemistry 48(11):2522–2528

    Article  CAS  Google Scholar 

  17. Ahmed SA, Byrne MP, Jensen M, Hines HB, Brueggemann E, Smith LA (2001) Enzymatic autocatalysis of botulinum A neurotoxin light chain. J Protein Chem 20(3):221–231

    Article  CAS  Google Scholar 

  18. Whitemarsh RC, Tepp WH, Bradshaw M, Lin G, Pier CL, Scherf JM, Johnson EA, Pellett S (2013) Characterization of botulinum neurotoxin A subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro. Infect Immun 81(10):3894–3902. doi:10.1128/IAI.00536-00513

    Article  CAS  Google Scholar 

  19. Pier CL, Chen C, Tepp WH, Lin G, Janda KD, Barbieri JT, Pellett S, Johnson EA (2011) Botulinum neurotoxin subtype A2 enters neuronal cells faster than subtype A1. FEBS Lett 585(1):199–206

    Article  CAS  Google Scholar 

  20. Torii Y, Kiyota N, Sugimoto N, Mori Y, Goto Y, Harakawa T, Nakahira S, Kaji R, Kozaki S, Ginnaga A (2011) Comparison of effects of botulinum toxin subtype A1 and A2 using twitch tension assay and rat grip strength test. Toxicon 57:93–99

    Article  CAS  Google Scholar 

  21. Mukai Y, Shimatani Y, Sako W, Asanuma K, Nodera H, Sakamoto T, Izumi Y, Kohda T, Kozaki S, Kaji R (2014) Comparison between botulinum neurotoxin type A2 and type A1 by electrophysiological study in healthy individuals. Toxicon 81:32–36. doi:10.1016/j.toxicon.2013.1012.1012

    Article  CAS  Google Scholar 

  22. Webb RP, Smith TJ, Wright P, Brown J, Smith LA (2009) Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit vaccines against toxin subtypes A1, A2, and A3. Vaccine 27(33):4490–4497

    Article  CAS  Google Scholar 

  23. Barash JR, Arnon SS (2014) A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins. J Infect Dis 209(2):183–191. doi:10.1093/infdis/jit1449

    Article  CAS  Google Scholar 

  24. Dover N, Barash JR, Hill KK, Xie G, Arnon SS (2014) Molecular characterization of a novel botulinum neurotoxin type H gene. J Infect Dis 209(2):192–202. doi:10.1093/infdis/jit1450

    Article  CAS  Google Scholar 

  25. Gonzalez-Escalona N, Thirunavukkarasu N, Singh A, Toro M, Brown EW, Zink D, Rummel A, Sharma SK (2014) Draft genome sequence of bivalent clostridium botulinum strain IBCA10-7060, encoding botulinum neurotoxin B and a new FA mosaic type. Genome Announc 2(6):e01275-01214. doi:10.1128/genomeA.01275-14

  26. Hill KK, Smith TJ, Helma CH, Ticknor LO, Foley BT, Svensson RT, Brown JL, Johnson EA, Smith LA, Okinaka RT, Jackson PJ, Marks JD (2007) Genetic diversity among botulinum neurotoxin-producing clostridial strains. J Bacteriol 189(3):818–832

    Article  CAS  Google Scholar 

  27. Tevell Aberg A, Bjornstad K, Hedeland M (2013) Mass spectrometric detection of protein-based toxins. Biosecur Bioterror 11(Suppl 1):S215–S226. doi:10.1089/bsp.2012.0072

    Article  Google Scholar 

  28. Kalb SR, Goodnough MC, Malizio CJ, Pirkle JL, Barr JR (2005) Detection of botulinum neurotoxin A in a spiked milk sample with subtype identification through toxin proteomics. Anal Chem 77(19):6140–6146

    Article  CAS  Google Scholar 

  29. Kalb SR, Baudys J, Rees JC, Smith TJ, Smith LA, Helma CH, Hill K, Kull S, Kirchner S, Dorner MB, Dorner BG, Pirkle JL, Barr JR (2012) De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics. Anal Bioanal Chem 403(1):215–226. doi:10.1007/s00216-00012-05767-00213

    Article  CAS  Google Scholar 

  30. Wang D, Baudys J, Rees J, Marshall KM, Kalb SR, Parks BA, Nowaczyk L 2nd, Pirkle JL, Barr JR (2012) Subtyping botulinum neurotoxins by sequential multiple endoproteases in-gel digestion coupled with mass spectrometry. Anal Chem 84:4652–4658

    Article  CAS  Google Scholar 

  31. Leveque C, Ferracci G, Maulet Y, Mazuet C, Popoff M, Seagar M, El Far O (2014) Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism. Biosens Bioelectron 57:207–212. doi:10.1016/j.bios.2014.02.015

    Article  CAS  Google Scholar 

  32. Mazuet C, Dano J, Popoff MR, Creminon C, Volland H (2010) Characterization of botulinum neurotoxin type A neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity. PLoS One 5(8), e12416

    Article  Google Scholar 

  33. Volland H, Lamourette P, Nevers MC, Mazuet C, Ezan E, Neuburger LM, Popoff M, Creminon C (2008) A sensitive sandwich enzyme immunoassay for free or complexed Clostridium botulinum neurotoxin type A. J Immunol Methods 330(1–2):120–129

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the toxicology department of DGA Maîtrise NRBC for its help with the animal experiments. This research was supported by grants from the Direction Générale de l’Armement and from Institut Pasteur.

Author information

Authors and Affiliations

  1. Direction Générale de l’Armement, DGA Maîtrise NRBC, 91710, Vert-le-Petit, France

    Valérie Morineaux, Didier Hilaire & Julien Enche

  2. Institut Pasteur, Unité des Bactéries anaérobies et Toxines, 75724, Paris, France

    Valérie Morineaux, Christelle Mazuet & Michel R. Popoff

Authors
  1. Valérie Morineaux
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christelle Mazuet
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Didier Hilaire
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julien Enche
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michel R. Popoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michel R. Popoff.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Morineaux, V., Mazuet, C., Hilaire, D. et al. Characterization of botulinum neurotoxin type A subtypes by immunocapture enrichment and liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 407, 5559–5570 (2015). https://doi.org/10.1007/s00216-015-8707-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-015-8707-1

Keywords

Search

Navigation