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

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. Graphical Abstract.

Keywords: BoNT; Botulinum neurotoxin; Botulism; Endopep-MS; Liver; Protease inhibitor.

Graphical Abstract

Fig. 1

Illustration of the BoNT-C cleavage site of the C peptide, and spectra of blank chicken serum (a) and chicken serum spiked with 5 MLD₅₀ BoNT-C (b), both analyzed by the regular Endopep-MS protocol

Fig. 2

Mass spectra of blank turkey liver (a) and turkey liver spiked with 10 MLD₅₀ BoNT-C (b) analyzed with the regular Endopep-MS protocol. The C peptide substrate is almost completely cleaved into m/z 2094.1 and 2250.2 in both the blank and the spiked sample, and in the spiked sample, only one of the expected BoNT-C cleavage products, m/z 1363.7, is visible. The peak at m/z 1543.8 represents the internal standard

Fig. 3

Results from method development, using the Endopep-MS protocol with addition of different volumes of protease inhibitor cocktail alone, or in combination with the salt washing step. Turkey liver homogenate samples were analyzed blank and spiked with BoNT-C or C/D and incubated for 21 h. The intensity of the unwanted cleavage products of the C substrate m/z 2250 and 2094 (illustrated in a and b, respectively), and the two expected peptide cleavage products for BoNT-C and C/D (illustrated in c and d)

Fig. 4

Results from method development of Endopep-MS for turkey liver. The samples were analyzed blank (a) and spiked with 10 MLD₅₀ of BoNT-C (b) by the modified Endopep-MS protocol. The peak at m/z 1543.8 represents the internal standard

Fig. 5

Results from method validation, using the regular and the modified Endopep-MS protocols, at both 3 and 21 h of incubation. Turkey liver homogenate samples were analyzed blank and spiked with BoNT-C, C/D, D, or D/C (six replicates each). The intensity of the unwanted cleavage products of the C substrate m/z 2250 and 2094 (illustrated in a and b, respectively), and the two expected peptide cleavage products for BoNT-C and C/D (illustrated in c and d) and for BoNT-D and D/C (illustrated in e and f)

Fig. 6

Spectrum of a clinical cattle liver sample (no. 3 in Table 2) analyzed by the modified Endopep-MS protocol. The C peptide substrate is almost completely consumed after 3-h incubation, cleaved into the two cleavage products significant for BoNT-C, i.e., m/z 1059.6 and 1363.6. The peak at m/z 1543.8 represents the internal standard