Research Article: Further optimization of peptide substrate enhanced assay performance for BoNT/A detection by MALDI-TOF mass spectrometry

Date Published: June 2, 2017

Publisher: Springer Berlin Heidelberg

Author(s): Dongxia Wang, Jakub Baudys, Kaitlin M. Hoyt, John R. Barr, Suzanne R. Kalb.


Rapid and sensitive detection of botulinum neurotoxins (BoNTs), which cause botulism, is essential in a public health emergency or bioterrorism event. We have previously developed a mass spectrometry (MS)-based functional method, Endopep-MS assay, for the fast detection and differentiation of all BoNT serotypes by affinity enriching the toxin and detecting the serotype-specific cleavage products of peptide substrates derived from the in vivo targets. To improve the performance of the Endopep-MS assay, we report here the further optimization of the peptide substrate for the detection of serotype A botulinum neurotoxins. An increased substrate cleavage was achieved by extending the original peptide N-terminus with optimized amino acid sequence, increasing the detection sensitivity of the method. In addition, the resistance of the substrate to nonspecific hydrolysis was dramatically improved by selectively substituting amino acids at the scissile bond and various other positions of the extended peptide. Moreover, incorporating the N-terminal hydrophobic residues dramatically improved the relative intensity of the cleavage products in the mass spectra. This allowed easy detection of the cleavage products, further enhancing the performance of the assay. The limit of detection for spiked serum sample was enhanced from 0.5 to 0.1 mouseLD50 and from 0.5 to 0.2 mouseLD50 for spiked stool.

Partial Text

Botulism is a life-threatening disease occurring in humans and animals caused by intoxication with potent bacterial neurotoxins, known as botulinum neurotoxins (BoNTs) [1–3]. BoNTs are expressed as a single protein of 150 kDa, consisting of two polypeptide chains linked through a disulfide bond. While the heavy chain is responsible for binding and entering target cells, the light chain cleaves at least one of three SNARE proteins in the peripheral neuron, subsequently blocking neurotransmitter release at the neuromuscular junction. BoNTs are classified into seven serotypes (A to G), depending on their antigenic properties and each serotype of BoNT includes several subtypes or variants. Human illness is associated with BoNTs of serotype A, B, E, and F. BoNT/A causes the most severe and longest lasting effects in humans followed by BoNT/B, /F, and /E. Due to their extreme toxicity, the ubiquitous nature of Clostridium botulinum spores in the environment, and the ease of preparation, BoNTs are likely agents for bioterrorism [4]. It is essential to have a rapid, simple, and sensitive method for the detection and quantification of botulinum toxins, for timely clinical confirmation of the disease state in botulism.

In this study, we further improved a peptide substrate used for BoNT/A detection in the Endopep-MS assay using a previously optimized substrate, Pep-1, as a template. Through the extension on the N-terminus of the peptide, a significant improvement on detection sensitivity was achieved. In addition, selective substitutions of amino acids at the scission bond and various other positions of the peptide sequence improved its resistance to nonspecific cleavage in biological matrices particularly in stool samples. Moreover, the incorporation of two N-terminal non-natural hydrophobic amino acids dramatically improved the relative intensities of the cleavage products in mass spectra to further enhance the performance of the optimal substrate in the assay. Furthermore, our study demonstrates that this new generation substrate is suitable for use as an efficient reagent for the detection of the subtypes of type A botulinum neurotoxin.




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