In-situ Detection of the Botulinum Neurotoxin A by Functionalized MALDI Chips Petra Darebna (Presenter) BioCeV

Authors: Petra Darebna(1,2), Petr Pompach (1,2), Jiri Dresler (3), Petr Novak (1,2)
(1) Institute of Microbiology, v.v.i., Czech Academy of Sciences, CZ (2) Faculty of Science, Charles University in Prague, CZ (3) Department of Microbiology and Biological Defence research, Military Health Institute, CZ

Botulinum neurotoxin (BoNT) is well known for causing the lethal disease botulism. The only widely accepted test for the identification of BoNTs in both clinical specimens and food is the mouse bioassay. In this study, we use MALDI chips functionalized by biotin-binding protein neutravidin prepared by ambient soft ion landing at atmospheric pressure for enrichment and detection of biotinylated peptides – products of specific endoproteinase activity of BoNT/A. The functionality of surfaces modified with biotin-binding molecules could help to drive the MALDI-TOF mass spectrometry toward its implementation into routine clinical practice.

Introduction

Botulinum neurotoxin (BoNT) is well known for causing the lethal disease botulism. The only widely accepted test for the identification of BoNTs in both clinical specimens and food is the mouse bioassay. There is a need for a rapid and sensitive method for detecting botulinum neurotoxins. Each BoNT type cleaves a unique site on proteins involved in neuronal transmission. Specific peptide substrates were designed by Barr lab to simulate these proteins for in vitro assays. The products of the enzymatic reaction are fragments of the peptide substrate, which are detectable by mass spectrometry. To enrich the peptide fragments from crude biological matrices, the peptide substrate is biotinylated at both termini. In this study we use MALDI chips functionalized with biotin-binding proteins to enrich and detect biotinylated peptide products of BoNT/A neurotoxin activity by MALDI-TOF mass spectrometry

Methods

The biotin-binding proteins neutravidin, streptavidin and avidin were diluted in 20mM ammonium bicarbonate buffer pH 7.8 to concentration of 10µM. The proteins were sprayed using lab-made apparatus for ion soft landing at atmospheric pressure. The dried ions were immobilized on polished indium tin oxide glass slides. The geometry of 16 spots array per chip was defined by adhesive foil mask with 2 mm spot diameter and 9.0 mm center-to-center distance. The MALDI chips were stored at 4°C before application of the samples.

BoNT/A was diluted with reaction buffer (5mM HEPES, 10µM ZnCl2, 10mM DTT, 1 mg/mL BSA, pH 7.3) at different concentration (from 200 ng/mL to 0.02 ng/mL). The N- and C-terminal biotinylated substrate peptide (KGSNRTRIDQGNQRATR-Nle-LGGK) was diluted with the same buffer to concentration of 20µM. The reaction was started by mixing the substrate peptide with BoNT/A toxin in 1:1 ratio. After 4 h incubation at 37°C, one microliter of reaction mixture was applied on spots of the functionalized MALDI chips. After 30 min incubation the chips were 3x washed with 20mM ammonium bicarbonate buffer pH 7.8 and 2x rinsed in water to remove residual salts. Each spot was covered with HCCA matrix. Enriched peptide substrate products were analyzed by Autoflex Speed MALDI-TOF mass spectrometer (Bruker Daltonics) in positive reflectron mode. The data were processed by flexAnalysis software (Bruker Daltonics).

Results

The performance of neutravidin/streptavidin/avidin-functionalized MALDI chips prepared by ambient ion landing to enrich biotinylated peptides was tested using biotinylated angiotensin standard followed by MALDI-TOF mass spectrometry. The MALDI chips functionalized by neutravidin provided highest yields of enriched peptides compared to streptavidin and avidin chips and were further used for detection of BoNT/A peptide products. Singly charged ions of biotinylated product peptides at m/z 1197.6 and 1699.8 were observed. The limit of detection was performed by dilution of BoNT/A in reaction buffer and monitoring of the 1197.6 peptide product. To lowest concentration of BoNT/A by which the peptide product at m/z 1197.6 was detected was 0.01 ng/mL.

Conclusions & Discussion

The functionalized MALDI chips modified by neutravidin achieve efficient peptide ionization to detect BoNT/A. The limit of detection for product peptide at m/z 1197.6 was 0.01 ng/mL of BoNT/A with signal to noise ratio 14.5. The limit for second product peptide observed at m/z 1699.8 was 0.1 ng/mL with S/N ratio 15.5. Comparing to other materials, non-reactive MALDI chips suffer minimal non-specific interactions with chemical species in the investigated sample. The whole procedure is fast, robust, high throughput and compatible with conventional mass spectrometers used in microbial laboratories.

This work was supported by Grant Agency of Charles University (1042217) and the Ministry of Education, Youth and Sports of CR within the LH15010, LD15089 and LQ1604 National Sustainability Program II (Project BIOCEV-FAR), the project "BIOCEV"; (CZ.1.05/1.1.00/02.0109) and Biovendor a.s. Research Agreement.