title: Subtyping botulinum neurotoxins by sequential multiple endoproteases in-gel digestion coupled with mass spectrometry.
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 serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.
authors.affiliation: National Center for Environmental Health, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia 30341, United States.
authors.forename: Dongxia
authors.initials: D
authors.lastname: Wang
authors.forename: Jakub
authors.initials: J
authors.lastname: Baudys
authors.forename: Jon
authors.initials: J
authors.lastname: Rees
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Marshall
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Kalb
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Parks
authors.forename: Louis
authors.initials: L
authors.lastname: Nowaczyk
authors.forename: James L
authors.initials: JL
authors.lastname: Pirkle
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D003014:Clostridium botulinum / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D005782:Gels; D019791:Guanidine / Q000737:chemistry; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D009498:Neurotoxins / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D040901:Proteomics; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry
chemical_list: D005782:Gels; D009498:Neurotoxins; D010450:Endopeptidases; D001905:Botulinum Toxins; D019791:Guanidine