Studies on botulinum neurotoxins type /C1 and mosaic/DC using Endopep-MS and proteomics

Authors

  • Hercules Moura,

    1. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention,Atlanta, GA
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  • Rebecca R. Terilli,

    1. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention,Atlanta, GA
    2. Association of Public Health Laboratories, Silver Spring, MD
    3. Oak Ridge Institute for Scientific Education, Oak Ridge, TN
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  • Adrian R. Woolfitt,

    1. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention,Atlanta, GA
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  • Maribel Gallegos-Candela,

    1. Battelle, Columbus OH, Working at Centers for Disease Control and Prevention
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  • Lisa G. McWilliams,

    1. Battelle, Columbus OH, Working at Centers for Disease Control and Prevention
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  • Maria I. Solano,

    1. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention,Atlanta, GA
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  • James L. Pirkle,

    1. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention,Atlanta, GA
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  • John R. Barr

    1. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention,Atlanta, GA
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  • Editor: Ake Forsberg

Correspondence: John R. Barr, Division of Laboratory Sciences, National Center for Environmental Health; Centers for Disease Control and Prevention, 4770 Buford Hwy NE. MS F-50 Atlanta, GA 30341, USA. Tel.: +1 770 488 7848; fax: +1 770 488 0509; e-mail: jbarr@cdc.gov

Abstract

Botulinum neurotoxins (BoNTs) are very potent toxins and category A biological threat agents. BoNT serotypes /C1 and /D affect birds and mammals and can be potentially lethal to humans. We have previously described the usefulness of the Endopep-MS method to detect the activity of BoNT A through G. This report was followed by the application of the method to clinical samples. The activity of the BoNT serotypes associated with human disease (/A, /B, /E, and /F) was successfully detected. However, BoNT/C and /D require different conditions for fast substrate cleavage, and a comprehensive description of a method to study BoNT/C and /D has not yet been reported. This work describes a new, optimized version of the Endopep-MS method to detect BoNTs /C1 and /DC either spiked directly in 20 μL of reaction buffer or spiked in a larger volume of buffer and further extracted using antibody-coated magnetic beads. It was found that the incubation temperature at 42 °C was more effective for both toxin serotypes, but each toxin serotype has an optimum cleavage pH. Additionally, we describe for the first time a proteomics study using a fast trypsin digestion method and label-free quantification of these toxin serotypes.

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