Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-2 (of 2 Records) |
Query Trace: Terilli RR[original query] |
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A historical and proteomic analysis of botulinum neurotoxin type/G
Terilli RR , Moura H , Woolfitt AR , Rees J , Schieltz DM , Barr JR . BMC Microbiol 2011 11 232 BACKGROUND: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. RESULTS: An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison. CONCLUSIONS: The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MSE data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin. |
Studies on botulinum neurotoxins type/C1 and mosaic/DC using endopep-MS and proteomics
Moura H , Terilli RR , Woolfitt AR , Gallegos-Candela M , McWilliams LG , Solano MI , Pirkle JL , Barr JR . FEMS Immunol Med Microbiol 2010 61 (3) 288-300 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 muL 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 degrees 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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