Abstract
Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release. Seven BoNT serotypes (A-G) exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid, mass spectrometry-based method (Endopep-MS) to detect and differentiate active BoNTs A, B, E, and F. This method uses the highly specific protease activity of the toxins with target peptides specific for each toxin serotype. The product peptides derived from the endopeptidase activities of BoNTs are detected by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In buffer, this method can detect toxin equivalents of as little as 0.01 mouse lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A high-performance liquid chromatography-tandem mass spectrometry method for quantifying active toxin, where the amount of toxin can be correlated to the amount of product peptides, is also described.
Highlights
Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release
We synthesized the specific portions of SNAP-25 and VAMP that are substrates for the 4 BoNT serotypes that commonly cause human botulism
We found an increase in the amount of BoNT-dependent cleavage products detected in the modified peptides
Summary
Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release. The product peptides derived from the endopeptidase activities of BoNTs are detected by matrix-assisted laserdesorption ionization time-of-flight mass spectrometry In buffer, this method can detect toxin equivalents of as little as 0.01 mouse lethal dose (MLD) and concentrations as low as 0.62 MLD50/mL. The mouse bioassay is the accepted standard and is the only widely accepted method for detecting BoNT [2,3,4,5] In this assay, mice receiving an intraperitoneal injection containing a sample with more than a minimum lethal dose show symptoms of botulinum intoxication and die [2,4]. The test was designed to detect and differentiate BoNT serotypes A, B, E, and F in a 1-day test and has the sensitivity of ≈10 mouse lethal dose (MLD)50/mL [5,6]. The ELISA is currently used primarily as a fast screening technique, and results are verified by the mouse bioassay [5]
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