Botulinum Neurotoxin Detection and Differentiation by Mass Spectrometry

Barr, John R.; Moura, Hércules; Boyer, Anne; Woolfitt, Adrian R.; Kalb, Suzanne R.; Pavlopoulos, Antonis J.; McWilliams, Lisa G.; Schmidt, Jürgen; Martinez, Rodolfo A.; Ashley, David L.

doi:10.3201/eid1110.041279

Cited by 159 publications

(150 citation statements)

References 14 publications

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“…The sample was previously determined to contain BoNT A by the Endopep-MS assay 22 and the mouse assay and was subjected to MESID to obtain further information on the toxin subtype and variant on protein level. The toxin was extracted with antibody-coated beads and three identical samples were loaded into a 1D SDS gel ( Figure S3).…”

Section: Subtyping Of a Bont-contaminated Carrot Juice Samplementioning

confidence: 99%

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-Gel Digestion Coupled with Mass Spectrometry

Wang¹,

Baudys²,

Rees³

et al. 2012

Anal. Chem.

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Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A through 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. But 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.

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Section: Subtyping Of a Bont-contaminated Carrot Juice Samplementioning

confidence: 99%

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-Gel Digestion Coupled with Mass Spectrometry

Wang¹,

Baudys²,

Rees³

et al. 2012

Anal. Chem.

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“…A floating consensus was derived as shown and was used to design two additional longer substrates (Table 1). LF-3 consists of the 11-amino acid core of LF-1 with an additional 3 amino (N)-terminal and 7 C-terminal amino acids that match the consensus, except for arginine (R 19 ), which was included for MS detection. LF-4 also includes the core, but extended sequences do not directly match the floating consensus.…”

Section: Resultsmentioning

confidence: 99%

Detection and Quantification of Anthrax Lethal Factor in Serum by Mass Spectrometry

et al. 2007

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The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.Anthrax is caused by infection with Bacillus anthracis, a sporeforming, Gram-positive bacterium. The dormant spore is resistant to extremes of temperature, desiccation, and a variety of chemical treatments. 1 The stability, ease of production, and infectious capacity of the spores confer B. anthracis with high potential as a biological weapon. 2 B. anthracis spores gain entry through a dermal abrasion or gastrointestinal lesion causing cutaneous or gastrointestinal anthrax, respectively, or are inhaled, causing pulmonary anthrax. Systemic infection from the progression of any of the three forms of anthrax frequently results in secondary shock, multiple organ failure, and death. 3 Early diagnosis is critical for effective treatment of pulmonary (inhalation) anthrax. 4 In the U.S. bioterrorism attacks of 2001, pulmonary anthrax had a 45% fatality rate despite antibiotic treatment and aggressive supportive care of the patients. 5,6 The two exotoxins of B. anthracis are binary combinations of protective antigen (PA) and either edema factor (EF) or lethal factor (LF). 7 The complex of PA and EF forms edema toxin (ETx) and PA complexed with LF forms lethal toxin (LTx). PA is secreted as an 83-kDa protein (PA83), which binds to known receptors TEM8 (tumor endothelium marker 8) 8 and CMG2 (capillary morphogenesis protein 2) 9 on the surface of target cells where it is cleaved by a furin-like cell surface protease to a 63-kDa protein (PA63). 10 PA83 may also be cleaved to the PA63 conformer by serum proteases. 11 Cleavage causes a conformational

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“…Several internal residues and the terminal structure of this peptide were modified from the corresponding sequence of the SNAP-25 185-260 based on previous reported study (Schmidt and Bostian 1997) and experience gained during the initial development of the assay (Barr, Moura et al 2005;Boyer, Moura et al 2005). To evaluate whether higher sensitivities of the assay could be accomplished by further optimization of this substrate, we re-examined its function and primary structure.…”

Section: Terminal Modificationsmentioning

confidence: 99%

“…To evaluate the performance of the new substrate in the assay for detecting BoNT/A present in complex samples, we evaluated the specific cleavage of Pep-21 using BoNT/A toxin spiked into four common sample matrices: serum, stool extract, cell culture supernatant, and milk. The assays were conducted following the general procedure of the Endopep-MS method for clinical samples (Barr, Moura et al 2005;Kalb, Moura et al 2006;Wang, Baudys et al 2011). The toxin was first enriched from a spiked matrix by BoNT/A-specific antibody immobilized on magnetic beads followed by the cleavage reaction in a solution containing toxin bound to beads, Pep-21 and other reaction components.…”

Section: Complex Biological Matrixmentioning

confidence: 99%

“…Similarly, a mass spectrometry (MS) based in vitro activity assay (Endopep-MS method) has been developed in our laboratory and has been the first in vitro method to prove effective in clinical samples such as serum and stool (scheme 1) (Barr, Moura et al 2005;Kalb, Garcia Rodriguez et al 2010;Boyer, Gallegos Candela et al 2011). In this method, affinity enriched BoNT was incubated with a peptide substrate derived from or mimicking the active-site containing region of a BoNT's natural target.…”

Section: Introductionmentioning

confidence: 99%

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Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

Wang

Baudys

et al. 2013

Analytical Biochemistry

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Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to man. 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 five fold with toxin spiked into buffer solution or different biological matrices.

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Botulinum Neurotoxin Detection and Differentiation by Mass Spectrometry

Abstract: A new rapid, mass spectrometry-based method to detect and differentiate botulinal neurotoxins is described.

Cited by 159 publications

References 14 publications

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-Gel Digestion Coupled with Mass Spectrometry

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-Gel Digestion Coupled with Mass Spectrometry

Detection and Quantification of Anthrax Lethal Factor in Serum by Mass Spectrometry

Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

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