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

National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
Analytical Biochemistry (Impact Factor: 2.22). 09/2012; 432(2). DOI: 10.1016/j.ab.2012.09.021
Source: PubMed


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.

Download full-text


Available from: Suzanne R. Kalb, Mar 27, 2015
  • Source
    • "e l s e v i e r . c o m / l o c a t e / y a b i o High sensitivity and a broad selectivity range have been reported for different BoNT serotypes tested in different media (buffer, food, serum, and stool) using the Endopep–MS assay [17] [18] [19] [20]. Recently, we reported on the design of a new peptide substrate for improved detection of BoNT type E (BoNT/E) [21]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNT's is achieved by the Endopeptidase-Mass-Spectrometry (Endopep-MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT/B in the Endopep-MS assay. Our strategy was based on reported BoNT/B-substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide has led to a 5 fold increased sensitivity of the assay both in buffer and in a clinically relevant, human spiked sera.
    Analytical Biochemistry 09/2014; 473. DOI:10.1016/j.ab.2014.09.016 · 2.22 Impact Factor
  • Source
    • "Endopep–MS is the first in vitro method proven to be effective for BoNT detection in clinical samples such as serum and stool. Endopep–MS assays with high selectivity and at different sensitivity ranges have been reported for all BoNTs [16] [17] [18] [19]. BoNT type E (BoNT/E) specifically cleaves SNAP-25 between residues Arg 180 and Ile 181 . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Botulinum neurotoxins (BoNTs) are the most toxic substances known to human. Endopep-MS is used as a specific and rapid in-vitro assay to detect BoNTs. In this assay, immuno-captured toxin cleaves a serotypespecific peptide-substrate and the cleavage-products are then detected by mass spectrometry. To further improve the sensitivity of the assay, we report here the rational design of a new substrate-peptide for the detection of BoNT/E. Our strategy was based on previously reported structural interactions, integrated with analysis method efficiency considerations. Integration of the newly designed substrate has led to a more than one-order of magnitude increased sensitivity of the assay.
    Analytical Biochemistry 04/2014; 456(1). DOI:10.1016/j.ab.2014.03.024 · 2.22 Impact Factor
  • Source
    • "Two different substrates were initially examined: a recombinant native substrate, fulllength SNAP-25, and a short peptide substrate, Peptide 1 (Scheme 1). Peptide 1 was derived and optimized from SNAP-25's C-terminal sequence surrounding the BoNT/A cleavage site and is used as the substrate in a mass spectrometry-based Endopep-MS assay [33]. The full-length subtype A1 and A2 were purified as a single neurotoxin protein while the subtype A5 was prepared as a complex including its associated proteins (Fig. 1, lanes 2–4). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening disease botulism through the inhibition of neurotransmitter release by cleaving essential SNARE proteins. There are seven serologically distinctive types of BoNTs and many subtypes within a serotype have been identified. BoNT/A5 is a recently discovered subtype of type A botulinum neurotoxin which possesses a very high degree of sequence similarity and identity to the well-studied A1 subtype. In the present study, we examined the endopeptidase activity of these two BoNT/A subtypes and our results revealed significant differences in substrate binding and cleavage efficiency between subtype A5 and A1. Distinctive hydrolysis efficiency was observed between the two toxins during cleavage of the native substrate SNAP-25 versus a shortened peptide mimic. N-terminal truncation studies demonstrated that a key region of the SNAP-25, including the amino acid residues at 151 through 154 located in the remote binding region of the substrate, contributed to the differential catalytic properties between A1 and A5. Elevated binding affinity of the peptide substrate resulted from including these important residues and enhanced BoNT/A5's hydrolysis efficiency. In addition, mutations of these amino acid residues affect the proteolytic performance of the two toxins in different ways. This study provides a better understanding of the biological activity of these toxins, their performance characteristics in the Endopep-MS assay to detect BoNT in clinical samples and foods, and is useful for the development of peptide substrates.
    Biochimica et Biophysica Acta 10/2013; 1834(12). DOI:10.1016/j.bbapap.2013.09.007 · 4.66 Impact Factor
Show more