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

Anal Biochem. 2013 Jan 15;432(2):115-23. doi: 10.1016/j.ab.2012.09.021. Epub 2012 Sep 24.


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.

MeSH terms

  • Acetylation
  • Amino Acid Sequence
  • Botulinum Toxins, Type A / analysis*
  • Botulinum Toxins, Type A / immunology
  • Botulinum Toxins, Type A / metabolism
  • Botulism / metabolism
  • Endopeptidases / metabolism*
  • Immunoglobulin G / immunology
  • Molecular Sequence Data
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*
  • Substrate Specificity


  • Immunoglobulin G
  • Peptides
  • Endopeptidases
  • Botulinum Toxins, Type A