Botulinum neurotoxin detection and differentiation by mass spectrometry.

Botulinum neurotoxins (BoNTs) are the most toxic substances known (1). They are produced under anaerobic conditions by strains of Clostridium botulinum, C. butyricum, and C. baratii (1). Intoxication with 1 of the 7 distinct serotypes of BoNT (A–G) causes botulism. One of 4 serotypes of BoNT (A, B, E, and F) is usually the cause of botulism in humans. BoNTs are zinc metalloproteases that cleave and inactivate specific cellular proteins essential for the release of the neurotransmitter acetylcholine (Figure 1). BoNT-A, -C, and -E cleave SNAP (synaptosomal-associated protein)-25; BoNT-B, -D, -F, and -G cleave synaptobrevin 2 (also called VAMP 2). Of the serotypes, only 1, BoNT-C, cleaves >1 site on a specific protein. In addition to cleaving SNAP-25, BoNT-C also cleaves syntaxin (1). Figure 1 Synaptobrevin on the synaptic vesicle must interact with syntaxin and SNAP (synaptosomal-associated protein)-25 on the neuronal membrane for fusion to occur, which allows the nerve impulse to be delivered across the synaptic junction. The botulinum neurotoxin ... Current methods for detecting BoNT include a mouse bioassay (2–4) and an enzyme-linked immunosorbent assay (ELISA) (5,6). The mouse bioassay is the accepted standard and is the only widely accepted method for detecting BoNT (2–5). 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). Many institutional animal care and use committees, including that of the Centers for Disease Control and Prevention, require mice to be euthanized after the onset of severe symptoms. In the mouse bioassay, when the injected dose is high, mice typically develop signs of botulism within 8 hours. At lower doses, mice are affected more slowly; hence, mice are observed for 4 days before a negative result is recorded. The mouse bioassay can also be used to differentiate BoNT serotypes (4). Mixtures of neutralizing antibodies are given to mice in conjunction with the sample. Mice receiving the appropriate anti-BoNT serotype antibody are asymptomatic and survive, while mice treated with the other serotype antibodies show symptoms of botulism (4). The mouse bioassay measures active toxin and is sensitive. The absolute amount of toxin detected in the mouse bioassay is not well defined but is thought to be 10–20 pg/mL for BoNT A (7,8). The main disadvantage of the mouse bioassay is that it requires euthanizing many animals. It also requires several days to determine the toxin level and type (4–6). Special animal facilities are also required, personal hazards are associated with injecting animals (5,6), and some clostridia produce nonbotulinal toxins that also kill mice (5). The ELISA is more rapid than the mouse bioassay, but it is not a functional assay. It recognizes protein antigenic sites and in general is somewhat less sensitive than the mouse bioassay (5,6). The ELISA was validated for detecting BoNT produced in cooked meat medium and tryptone peptone glucose yeast extract (TPGY). 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). In a recent study, the ELISA performed well in most laboratories at the 100 MLD50/mL and 10,000 MLD50/mL levels (5). In this study, some cross-reactivity occurred among BoNT cultures and with nonbotulinum cultures (5). At 100 MLD50/mL, a >7% false-negative rate in TPGY was observed; at 10,000 MLD50/mL, a 1.5% false-positive rate for BoNT-A and a 28.6% false-positive rate for BoNT-F occurred (5). The ELISA is currently used primarily as a fast screening technique, and results are verified by the mouse bioassay (5). Several in vitro assays have been developed to detect the activities of the different BoNT serotypes (7–14). This approach has led to methods that are based on the natural substrates that are cleaved by the BoNTs and use fluorescence to detect toxin activity. These types of assays are >2 orders of magnitude less sensitive than the mouse bioassay and have not been proven for use with environmental, food, or clinical samples. They are also prone to giving false-positive results in samples that contain proteases because the specific site of cleavage cannot easily be determined in a fluorescence-based assay (15). Another method that combines immunoaffinity chromatography with specific antibodies for cleavage products has been successful in detecting BoNT B in some foods at lower detection limits than the mouse bioassay (10). We introduce here the concept and preliminary data on a new, rapid, mass spectrometry–based, functional method for detecting, differentiating, and quantifying 4 BoNT serotypes. This method is based on both the unusual endopeptidase activities of these enzymes and specific detection of the unique peptide products by mass spectrometry (Endopep-MS). Because each BoNT serotype has a unique cleavage site on a unique peptide, the mass-specific product peptides detected by mass spectrometry differentiate active BoNT serotypes. Substrate peptides that are specific for each serotype are incubated with BoNT; then serotype-specific product peptides are detected by either matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or by high-performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry (HPLC-ESI/MS/MS). Thus, this method combines the biologic specificity of the BoNT enzymatic activity with the unparalleled detection specificity of mass spectrometry.