Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples
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RESEARCH PAPER
Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples with application to samples collected during animal botulism outbreaks Annica Tevell Åberg 1,2 & Ida Karlsson 1,2 & Mikael Hedeland 1,2 Received: 21 July 2020 / Revised: 29 September 2020 / Accepted: 9 October 2020 # The Author(s) 2020
Abstract Botulinum neurotoxins (BoNTs) are the most potent toxins known and they cause the paralytic disease botulism in humans and animals. In order to diagnose botulism, active BoNT must be detected in biological material. Endopep-MS is a sensitive and selective method for serum samples, based on antibody capture, enzymatic cleavage of target peptides, and detection of cleavage products using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In many cases of animal botulism, serum samples are not available or they do not contain detectable amounts of BoNT and liver sampling is an alternative for postmortem examinations. However, the Endopep-MS method is impaired by the inherent protease activity of liver samples. In the presented study, the Endopep-MS method has been successfully modified and validated for analysis of cattle, horse, and avian liver samples, introducing a combination of a salt washing step and a protease inhibitor cocktail. These modifications resulted in a substantial decrease in interfering signals and increase in BoNT-specific signals. This led to a substantial improvement in sensitivity for especially BoNT-C and C/D which are among the most prominent serotypes for animal botulism. Botulism was diagnosed with the new method in liver samples from dead cattle and birds from outbreaks in Sweden. Keywords Endopep-MS . Botulism . Botulinum neurotoxin . BoNT . Liver . Protease inhibitor
Introduction The paralytic disease botulism is caused by botulinum neurotoxins (BoNTs) which are produced by anaerobic bacteria, mainly Clostridium botulinum. There are several known serotypes of BoNTs which are denoted A, B, C, D, E, F, G, and X [1–3]. All of the BoNTs are around 150 kDa large proteins
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-03001-z) contains supplementary material, which is available to authorized users. * Mikael Hedeland [email protected] 1
Department of Chemistry, Environment, and Feed Hygiene, National Veterinary Institute (SVA), 751 89 Uppsala, Sweden
2
Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Faculty of Pharmacy, Uppsala University, P.O. Box 574, 751 23 Uppsala, Sweden
that consist of a heavy chain, responsible for the transport across the neuronal membrane into the nerve cell, and a light chain, responsible for the toxic zinc metalloprotease activity. Inside the nerve cell, the light chain of BoNT cleaves one of the three proteins essential for the soluble N-ethylmaleimidesensitive factor activating protein receptor (SNARE) complex formation, resulting in inhibition of the rele
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