Subtyping botulinum neurotoxins by sequential multiple endoproteases in-gel digestion coupled with mass spectrometry.

Analytical Chemistry
Dongxia WangJohn R Barr

Abstract

Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A-G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. However, with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult, and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotype...Continue Reading

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Citations

Sep 18, 2014·Analytical and Bioanalytical Chemistry·Kristian BjörnstadMikael Hedeland
Aug 12, 2014·Applied and Environmental Microbiology·K A WeedmarkC R Corbett
Aug 1, 2013·Reviews in Analytical Chemistry·Suzanne R Kalb, John R Barr
Feb 12, 2021·Archives of Toxicology·Osnat RosenRan Zichel

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