Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances
known to humankind. It is essential to have a simple, quick, and sensitive method for the detection
and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory
has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and
differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique
cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of
seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and
animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel
peptide substrates for the detection of BoNT/E activity through systematic and comprehensive
approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement
in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and
trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD50/ml was achieved using
the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum,
stool, and food samples.
Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances
known to humankind. It is essential to have a simple, quick, and sensitive method for the detection
and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory
has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and
differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique
cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of
seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and
animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel
peptide substrates for the detection of BoNT/E activity through systematic and comprehensive
approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement
in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and
trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD50/ml was achieved using
the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum,
stool, and food samples.
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