title: Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep-MS assay.
abstract: 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.
authors.affiliation: Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
authors.forename: Dongxia
authors.initials: D
authors.lastname: Wang
authors.affiliation: Battelle Memorial Institute under contract at Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
authors.forename: Joan
authors.initials: J
authors.lastname: Krilich
authors.affiliation: Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
authors.forename: Jakub
authors.initials: J
authors.lastname: Baudys
authors.affiliation: Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
authors.affiliation: Centers for Disease Control and Prevention, Atlanta, GA 30341, USA. Electronic address: skalb@cdc.gov.
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Kalb
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001681:Biological Assay / Q000379:methods; D001905:Botulinum Toxins / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D007928:Lethal Dose 50; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods / Q000706:statistics & numerical data; D051379:Mice; D009498:Neurotoxins / Q000032:analysis* / Q000633:toxicity; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D009498:Neurotoxins; D010455:Peptides; D014357:Trypsin; D001905:Botulinum Toxins; C096326:botulinum toxin type E
keywords: Botulinum neurotoxin; Botulism; Mass spectrometry; Peptide substrate