Enhanced detection of type C botulinum neurotoxin by the Endopep-MS assay through optimization of peptide substrates

It is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum neurotoxins, the most toxic substances and the causative agents of botulism. Type C botulinum neurotoxin (BoNT/C) represents one of the seven members of distinctive BoNT serotypes (A to G) that cause botulism in animals and avians. Here we report the development of optimized peptide substrates for improving the detection of BoNT/C and /CD mosaic toxins using an Endopep-MS assay, a mass spectrometry-based method that is able to rapidly and sensitively detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Based on the sequence of a short SNAP-25 peptide, we conducted optimization through a comprehensive process including length determination, terminal modification, single and multiple amino acid residue substitution, and incorporation of unnatural amino acid residues. Our data demonstrate that an optimal peptide provides a more than 200-fold improvement over the substrate currently used in the Endopep-MS assay for the detection of BoNT/C1 and /CD mosaic. Using the new substrate in a four-hour cleavage reaction, the limit of detection for the BoNT/C1 complex spiked in buffer, serum and milk samples was determined to be 0.5, 0.5 and 1mouseLD50/mL, respectively, representing a similar or higher sensitivity than that obtained by traditional mouse bioassay.