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

Abstract

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.

Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum

“…The water was removed and 20.0 μL of reaction buffer (10 mM DTT, 200 μM ZnCl 2 , 1.0 mg/mL BSA, and 50 μM substrate peptide in 20 mM HEPES buffer pH 7.3) was added. Since the first Endopep-MS article published by Boyer et al [43], the substrate peptides used for the different BoNT serotypes have been continuously improved [44–48,39,38]. The different substrate peptides that were used in this study are listed in Table 2.…”

“…The selectivity of the Endopep-MS is achieved on two levels, 1) by using serotype specific antibodies directed towards the BoNTs, and 2) by using a specific substrate for each serotype. The sensitivity has been proven to be as good as, or better, than that of the MBA [38,39]. Furthermore, Endopep-MS detects active toxin down to serotype level.…”

Three Enzymatically Active Neurotoxins of Clostridium botulinum Strain Af84: BoNT/A2, /F4, and /F5

“…Peptide substrates Acetyl-RGSNKPKIDAGNQRATRXLGGR-NH 2 (abbreviated as SubA) for assessment of BoNT/A activity (where X is norleucine) and TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL for assessment of BoNT/F activity (abbreviated as SubF) were synthesized by Midwest Bio-tech Inc. (Fishers, IN). The peptide substrate for BoNT/A has been modified from the original sequence of SNAP-25 to produce a more efficient substrate for BoNT/A cleavage 24 .…”

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

“…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].…”