title: Studies on botulinum neurotoxins type /C1 and mosaic/DC using Endopep-MS and proteomics.
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.
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Hercules
authors.initials: H
authors.lastname: Moura
authors.forename: Rebecca R
authors.initials: RR
authors.lastname: Terilli
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Woolfitt
authors.forename: Maribel
authors.initials: M
authors.lastname: Gallegos-Candela
authors.forename: Lisa G
authors.initials: LG
authors.lastname: McWilliams
authors.forename: Maria I
authors.initials: MI
authors.lastname: Solano
authors.forename: James L
authors.initials: JL
authors.lastname: Pirkle
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000032:analysis*; D010450:Endopeptidases / Q000032:analysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D014116:Toxicology / Q000379:methods*
chemical_list: C096325:botulinum toxin type D; D010450:Endopeptidases; D001905:Botulinum Toxins; C096324:botulinum toxin type C