title: Improved detection of botulinum neurotoxin serotype A by Endopep-MS through peptide substrate modification.
abstract: Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep-MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep-MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices.
authors.affiliation: National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
authors.forename: Dongxia
authors.initials: D
authors.lastname: Wang
authors.forename: Jakub
authors.initials: J
authors.lastname: Baudys
authors.forename: Yiming
authors.initials: Y
authors.lastname: Ye
authors.forename: Jon C
authors.initials: JC
authors.lastname: Rees
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
authors.forename: James L
authors.initials: JL
authors.lastname: Pirkle
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Kalb
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D019274:Botulinum Toxins, Type A / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D001906:Botulism / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D007074:Immunoglobulin G; D010455:Peptides; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A