Open AccessStudies on botulinum neurotoxins type /C1 and mosaic/DC using Endopep‐MS and proteomics
Author(s) -
Moura Hercules,
Terilli Rebecca R.,
Woolfitt Adrian R.,
GallegosCandela Maribel,
McWilliams Lisa G.,
Solano Maria I.,
Pirkle James L.,
Barr John R.
Publication year - 2011
Publication title -
fems immunology & medical microbiology
Language(s) - English
Resource type - Journals
eISSN - 1574-695X
pISSN - 0928-8244
DOI - 10.1111/j.1574-695x.2010.00774.x
Subject(s) - toxin , serotype , biology , trypsin , clostridium botulinum , botulinum toxin , microbiology and biotechnology , proteomics , chromatography , virology , biochemistry , chemistry , enzyme , neuroscience , gene
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.