Identifying Botulism Toxins

In a new study, researchers have developed a new way to analyse the deadly protein botulinum neurotoxin. This discovery makes it possible to differentiate its numerous subtypes, many of which differ by only a few amino acids. Botulinum toxin induces paralysis and is considered a potential bioterrorism agent. Subtype identification could be particularly important in tracking down the sources of outbreaks of botulism, the illness caused by the toxin. Not only does the toxin have seven serotypes, designated A through G, but those serotypes themselves contain numerous subtypes and variants. Methods based on the polymerase chain reaction can identify the toxin subtypes, but they require DNA from the toxin-producing organisms, which include Clostridium botulinum. In many cases, only the toxin itself is available. By expanding a traditional analytical method, which involves enzymatically digesting the large protein in a gel and then sequencing the resulting peptides via mass spectrometry, research chemists John R. Barr of the Centres for Disease Control & Prevention and colleagues were able to get their results. Barr says the traditional method is inefficient because of incomplete digestion, and it often fails to produce enough peptides to generate a thorough sequence identification. Instead, the group used several enzymes in sequence in a single gel to chop the toxin into many more peptides. The sequential digestions in the same gel band allow the team to systematically detect more thoroughly digested protein pieces, yielding dramatically higher sequence coverage, Barr says. Analysis of the resulting peptides by mass spectrometry made it possible for them to accurately identify more than 98% of the protein sequence of a botulinum toxin variant in contaminated carrot juice. The method could be generalised “to identify or distinguish other important protein toxins, or any proteins with variants that have close sequence identities,” notes Luisa W. Cheng, a USDA biologist.

Chemical & Engineering News, 21 May 2012 ; ;