Novel activity assay for botulotoxin A1 detection using functionalized chips and matrix-assisted laser desorption/ionization mass spectrometry
Year: 2025
Type: article
Abstract: Background Botulinum neurotoxins (BoNTs) are a group of neurotoxins produced by Clostridium bacteria. Their effect on neuro-muscular connections through cleaving proteins of the SNARE complex results in blocking acetylcholine signal transduction. The FDA-approved mouse bioassay, which involves exposing live mice to potentially contaminated food, is the most widely used method. However, this assay is costly, time-consuming, and raises ethical concerns. Therefore, there is a need for alternative assays that can enzymatically measure the activity of BoNTs.Research design and methods We present an approach that combines the EndoPep-MS assay with protein affinity chips fabricated using ion soft-landing technology. Toxic activity is indirectly assessed by monitoring the N- and C-terminal fragments of the substrate peptide. This new method employs a protein array with affinity molecules targeting either the BoNT/A1 or the substrate peptide. Both variants enable in-situ reaction and detection of substrate peptides via MALDI-ToF MS on the protein chip.Results This method demonstrated successful detection of active BoNT/A1 in both buffer and complex matrices, achieving a detection limit of 0.5 ng/mL.Conclusions This study reports the in-situ detection of botulotoxin A1 using functionalized MALDI chips. The advantages of the MALDI chip technology include speed, robustness, cost-effectiveness, and possible automatization.
Source: Expert Review of Proteomics
Authors: Zuzana Kalaninova, Josef Dvorak, Jiri Dresler, Michael Volny, Petr Novak, Petr Pompach
Language: English
Subfield: Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Funders: Health Research, Univerzita Karlova v Praze, Grantová Agentura, Univerzita Karlova, Technology Agency of the Czech Republic, European Commission, Ministerstvo Školství, Mládeže a Tělovýchovy, Akademie Věd České Republiky, Agentura Pro Zdravotnický Výzkum České Republiky, European Regional Development Fund, Mikrobiologický Ústav, Akademie Věd České Republiky
Awards: G1003250212, G176604203, G1822242985, G1935710221, G1940663492, G2343156113, G2486988760, G2534379819, G2537698827, G291036901, G3011267836, G308298343, G3490171961, National institute for Neurological Research, G3622461675, G3735356012, G3810236772, G3858648280, G4280972364, G4396925471, National centre for new methods of diagnosis, monitoring, treatment and prevention of genetic diseases, G5007093561, G5597571815, G5641063287, G5916014575, G5918706011, G6761430300, G7534432959, G7662032182, G7890394945, G8140083606, Czech Infrastructure for Integrative Structural Biology, G869252295
OpenAlex ID: W4408624655