From the Mouse to the Mass Spectrometer:  Detection and Differentiation of the Endoproteinase Activities of Botulinum Neurotoxins A−G by Mass Spectrometry

Boyer, Anne; Moura, Hércules; Woolfitt, Adrian R.; Kalb, Suzanne R.; McWilliams, Lisa G.; Pavlopoulos, Antonis J.; Schmidt, Jürgen; Ashley, David L.; Barr, John R.

doi:10.1021/ac050485f

Cited by 132 publications

(123 citation statements)

References 33 publications

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“…A method using mass spectrometry (MS) for the detection of the SNARE cleavage products has been developed at the US CDC; the assay has been called Endopep-MS assay [126,127]. Mass spectrometry in tandem with liquid chromatography can be used not only for the quantification of the toxin, but it is also able to differentiate all seven toxin serotypes by discriminating between each serotype’s unique cleavage product(s).…”

Section: Endopep-msmentioning

confidence: 99%

“…Mass spectrometry in tandem with liquid chromatography can be used not only for the quantification of the toxin, but it is also able to differentiate all seven toxin serotypes by discriminating between each serotype’s unique cleavage product(s). The detection limits achieved a range from 0.039 to 0.625 MLD 50 /mL for BoNT/A, /B, /E and /F in a buffer [126]. However, the sensitivity of the method in serum and stool samples was not as impressive.…”

Section: Endopep-msmentioning

confidence: 99%

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Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Čapek

Dickerson

2010

Toxins

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Sensitive and rapid detection of botulinum neurotoxins (BoNTs), the most poisonous substances known to date, is essential for studies of medical applications of BoNTs and detection of poisoned food, as well as for response to potential bioterrorist threats. Currently, the most common method of BoNT detection is the mouse bioassay. While this assay is sensitive, it is slow, quite expensive, has limited throughput and requires sacrificing animals. Herein, we discuss and compare recently developed alternative in vitro detection methods and assess their ability to supplement or replace the mouse bioassay in the analysis of complex matrix samples.

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Section: Endopep-msmentioning

confidence: 99%

Section: Endopep-msmentioning

confidence: 99%

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Čapek

Dickerson

2010

Toxins

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“…These IgGs have been used in a previous study (Moura et al, 2008). Peptides were synthesized by Los Alamos National Laboratory, and the sequences of the substrates and the cleavage products were the same as the ones described by Boyer et al (2005), with the addition of peptides 128 and 171 (see Table 1) for increased certainty in the detection of BoNT/C1. Dynabeads Protein G were purchased from Invitrogen at 1.3 g ml 21 in PBS (pH 7.4) containing 0.1 % Tween 20 (PBST) and 0.02 % sodium azide.…”

Section: Methodsmentioning

confidence: 99%

“…In 2005, another concept was reported, based on BoNT cleavage of synthetic peptides followed by detection of the product peptides by matrix-assisted laser desorption ionization (MALDI) and/or electrospray ionization (ESI) mass spectrometry (MS) Boyer et al, 2005). The method, denoted Endopep-MS, was proven to be successful for detection of all seven BoNT serotypes in buffer solution, and the detection limits for BoNT/A, /B, /E and /F were lower than those obtained by the mouse bioassay.…”

Section: Introductionmentioning

confidence: 99%

Confirmation of botulism in birds and cattle by the mouse bioassay and Endopep-MS

Hedeland

Moura

Båverud

et al. 2011

Journal of Medical Microbiology

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There have been several outbreaks of botulism among poultry and wild birds in Sweden in recent years. The National Veterinary Institute of Sweden (SVA) has identified botulinum neurotoxin (BoNT)/C1 or the mosaic BoNT/C1D using the mouse bioassay. This is believed to be the first report on the application of the Endopep mass spectrometry (Endopep-MS) method to selected clinical animal (serum and liver) samples and a feed sample that had previously given positive test results with the mouse bioassay. In the mouse bioassay eight of the eleven samples were found to be neutralized by both BoNT/C1 and /D antitoxins; the other three were neutralized only by BoNT/C1 antitoxin, but the mice showed a prolonged survival time when the samples had been treated with /D antitoxin. The Endopep-MS analysis, on the other hand, demonstrated only BoNT/C1 activity for all eleven samples. This suggests that at least eight of the samples were of the chimeric toxin type BoNT/C1D, where the enzymically active site is identical to that of BoNT/C1, while other parts of the protein contain sequences of BoNT/D. This is the first step of a cross-validation between the established mouse bioassay and the Endopep-MS of serotypes BoNT/C1 and /C1D. Endopep-MS is concluded to have potential as an attractive alternative to the mouse bioassay.

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“…Several internal residues and the terminal structure of this peptide were modified from the corresponding sequence of the SNAP-25 185-260 based on previous reported study (Schmidt and Bostian 1997) and experience gained during the initial development of the assay (Barr, Moura et al 2005;Boyer, Moura et al 2005). To evaluate whether higher sensitivities of the assay could be accomplished by further optimization of this substrate, we re-examined its function and primary structure.…”

Section: Terminal Modificationsmentioning

confidence: 99%

Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

Wang

Baudys

et al. 2013

Analytical Biochemistry

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Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to man. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep-MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype specific antibodies and detecting the unique and serotype specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep-MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity five fold with toxin spiked into buffer solution or different biological matrices.

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From the Mouse to the Mass Spectrometer: Detection and Differentiation of the Endoproteinase Activities of Botulinum Neurotoxins A−G by Mass Spectrometry

Cited by 132 publications

References 33 publications

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Confirmation of botulism in birds and cattle by the mouse bioassay and Endopep-MS

Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

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