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Novel activity assay for botulotoxin A1 detection using functionalized chips and matrix-assisted laser desorption/ionization mass spectrometry

ABSTRACT: 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. To prevent external intoxication by BoNT, analysis of potential sources of BoNT needs to be performed. Currently available methods target the presence of BoNTs molecules but only a fraction is screening real toxic activity. The FDA-approved mouse bioassay, based on exposing live mice to the potentially contaminated food, is the most common. Unfortunately, this assay is expensive, time consuming and ethically questionable. That is why there is a need for assays that can measure the activity of BoNTs enzymatically. We present an assay combining known EndoPep-MS assay with detection protein affinity chips manufactured by ion soft-landing technology. The toxic activity is indirectly determined by monitoring their endopeptidase activity as a change in molecular mass of cleaved substrate peptides exposed to BoNTs. Our modification utilizes a protein array modified by affinity molecules towards either the BoNTs or the substrate peptides. Both variants allow to perform the in-situ reaction and detection of substrate peptides by MALDI-ToF MS on the protein chip. This method demonstrated successful detection of active BoNT/A1 in both buffer and complex matrices, achieving a detection limit of 0.5 ng/mL.

INSTRUMENT(S): autoflex

ORGANISM(S): Clostridium Botulinum

SUBMITTER: Zuzana Kalaninova

LAB HEAD: Petr Pompach

PROVIDER: PXD058240 | Pride | 2025-03-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	BoNTA-A1688-buffer-results.csv	Csv
	BoNTA-A1688-buffer.zip	Other
	BoNTA-A1688-food-results.csv	Csv
	BoNTA-A1688-food.zip	Other
	BoNTA-HcA78-buffer-results.csv	Csv

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Project description:Matrix-Assisted Laser Desorption/Ionization Time of Flight mass spectrometry (MALDI-TOF MS) has become a standard clinical tool for microbial identification. Its utilization in clinical microbiology laboratories keeps expanding from basic identifications towards detecting antibiotic resistance and microbial toxins. Clostridiodes difficile represents an emerging bacterial pathogen causing severe gastrointestinal infection associated with antibiotic therapy affecting normal gut microbiota. The virulence of C. difficile is mainly caused by two toxins, namely Toxin A and Toxin B. Established diagnostic methods, including PCR and enzyme-linked immunoassays, detect either the presence of the microorganism itself or the presence and concentration of the toxins, with only limited ability to gauge infection severity and evaluate the actual biochemical activity of the toxins and their potency to cause harm. This work addresses the infection severity using the functional activity detection of C. difficile toxins by MALDI-TOF MS assay performed on affinity MALDI chips. The MALDI chips are modified using ambient ion landing technology with an array of immobilized Neutralite Avidin (NeutrAvidin) spots that allow secondary immobilization of biotin-tagged recombinant RhoA, and this protein is a substrate for the Clostridiodes difficile toxin’s enzymatic activity. Monitoring mass shift due to the in-vitro glycosylation of the RhoA in the MALDI-TOF mass spectrum provides information about the Toxin’s B activity in the sample. The assay was used to analyze 20 samples of patients suspected of C. difficile infection and compared with the results obtained by standard methods. Observing the Toxin B activity, rather than the concentration, enhances diagnostic efficiency and offers valuable information about infection potency without the extensive resources required by reference methods like stool cytotoxicity assay and toxigenic culture. The presented assay is a representative example of analytical chemistry helping clinical microbiology achieve modern symptom-based diagnostics.

Project description:For a long time, Neanderthals were considered hunters of large mammals, whereas the diversification of the exploited faunal spectrum to include smaller taxa, including birds, was assumed to be specific to anatomically modern humans. In recent decades, archaeozoological analyses of faunal remains from layers associated with Middle Palaeolithic lithic industries have revealed traces of human manipulation of small taxa, indicating the exploitation of a wider range of animals than previously thought, including small or fast-moving animals such as molluscs, leporids and birds. These new data have challenged the view that Neanderthals did not exploit small animals, thereby narrowing the behavioral gap with anatomically modern humans. Nevertheless, the information currently available comes almost exclusively from southern Europe and the nature of Neanderthal small fauna exploitation in northern Europe remains largely unknown. The present study aims to fill this gap by applying archaeozoological methods, including detailed taphonomic and traceological analyses, to 118 bird remains recovered from levels containing Middle Palaeolithic industries at Scladina cave, southern Belgium. Analyses of proteomics were applied to clarify the taxonomic identity of two morphologically non-diagnostic elements. Compared to mammal remains, bird bones, most of which belong to the order Galliformes, are scarce at Scladina Cave. This is likely due to conservation bias. Traces of non-human predators or scavengers, suggest that mammalian carnivores are responsible for accumulating a considerable portion of the avian assemblage. In total, seven bird bones exhibit anthropogenic traces, and one element presents questionable traces. Various Galliformes and a cormorant were exploited likely for their meat, during MIS 5 and/or 6 and MIS 6. The terminal posterior phalanx (talon) of a raptor of the size of a pomarine eagle displays intense polishing that could be linked to human manipulation of this element (MIS 5 and/or 6), although in the absence of tool marks this remains hypothetical at this stage. On the radius of a Western capercaillie, two deep incisions may indicate bone working, and intense use-wear on one of the fractured ends indicates that the bone has been utilized, potentially on soft organic material (MIS 6). This study provides the first evidence of the exploitation of birds during the Middle Palaeolithic in Belgium and constitutes the only detailed archaeozoological analysis of bird material in northwestern Europe. The likely transformation and use of a bird bone is only the second example recovered from Neanderthal occupations. The novel taxa identified as Neanderthal prey highlight the plasticity of Neanderthal ecological behavior, adapting to different landscapes and climates and exploiting the full spectrum of locally available prey.

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Novel activity assay for botulotoxin A1 detection using functionalized chips and matrix-assisted laser desorption/ionization mass spectrometry

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