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

Abstract

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. Graphical abstractMass spectra of optimized and old peptide substrates with BoNT/A

AbobotulinumtoxinA (Dysport®), OnabotulinumtoxinA (Botox®), and IncobotulinumtoxinA (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients

“…In our hands, however, MALDI-TOF was not sensitive enough for these samples using the substrate peptide described in Kalb et al [26]. Recently, Wang et al [32] published a paper with peptide substrate sequences that improve the sensitivity of the detection of BoNT/A. At the time that the Wang et al paper appeared, however, we were already conducting studies with the CE-LIF method, and the substrate used provided the necessary sensitivity for this purpose.…”

“…At the time that the Wang et al paper appeared, however, we were already conducting studies with the CE-LIF method, and the substrate used provided the necessary sensitivity for this purpose. In future studies it may be worth assessing the further optimised substrate peptides mentioned in [32] with MALDI-TOF detection as well. Alternatively, the samples could be analysed with liquid chromatography–mass spectrometry.…”

A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices

“…In this study, we have established that CANARY ® can detect BoNT/A holotoxin with a LOD of 10.0 ± 2.5 ng/mL. This LOD is within the range of lateral flow devices but not as sensitive as other assays [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. An ultrasensitive gold nanoparticle endopeptidase-based lateral flow has been developed and has a LOD ≈ 20.0 pg/mL in sera comparable to the mouse bioassay; however, this assay requires 12 h of digestion but food and beverage matrices were not evaluated [ 28 ].…”

“…BoNT detection assays utilize multiple methods including antibody-based, mass-spectrometry, nucleic acid-based, cell-based, and enzymatic assays, as well as in vivo and ex vivo mouse assays in buffer and some matrices [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. All of the current technologies have advantages and disadvantages.…”

“…Additionally, newer assays have combined different technologies to improve the sensitivity of detection. Depending on the assay, the detection limits range from sub-picogram to nanogram per mL or attomolar to pM for buffer and some food matrices [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ].…”