Botulinum neurotoxin detection and differentiation by mass spectrometry

Abstract

Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release. Seven BoNT serotypes (A-G) exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid, mass spectrometry-based method (Endopep-MS) to detect and differentiate active BoNTs A, B, E, and F. This method uses the highly specific protease activity of the toxins with target peptides specific for each toxin serotype. The product peptides derived from the endopeptidase activities of BoNTs are detected by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In buffer, this method can detect toxin equivalents of as little as 0.01 mouse lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A high-performance liquid chromatography-tandem mass spectrometry method for quantifying active toxin, where the amount of toxin can be correlated to the amount of product peptides, is also described.

Figure 1

Synaptobrevin on the synaptic vesicle must interact with syntaxin and SNAP (synaptosomal-associated protein)-25 on the neuronal membrane for fusion to occur, which allows the nerve impulse to be delivered across the synaptic junction. The botulinum neurotoxin serotypes cleave the peptide bonds at specific sites on the 3 proteins, as indicated. Cleavage of any 1 of these proteins prevents vesicle membrane docking and nerve impulse transmission.

Figure 2

Substrate peptide sequences, the botulinum neurotoxin (BoNT) serotype predicted cleavage product sequences, and masses of the substrate and product peptides. Peptides for BoNT-A and -E were derived from the human SNAP (synaptosomal-associated protein)-25 protein. The substrate peptide for BoNT A, 187-SNKTRIDEANQRATKML-203, was modified to biotin(ε)-KG(K189->R and K201->R)GGK-(ε)Biotin. The BoNT-E substrate sequence was also from human SNAP-25 (156–186). Substrate peptides for BoNT-B and -F are from human synaptobrevin 2; the BoNT-B substrate (3) is from 59–93 in the sequence and the BoNT-F substrate is from 35–74.

Figure 3

High-performance liquid chromatography–electrospray ionization-tandem mass spectrometry chromatogram showing the botulinum neurotoxin (BoNT)-A substrate and product ions (CT, C-terminal; NT, N-terminal) from a reaction with 25 mouse lethal dose (MLD)50 BoNT-A. Each peptide has both a quantification ion (top trace) and a verification ion (lower trace). Isotopically labeled standards are added (traces not shown) as internal standards for quantification. The labeled peptides co-elute with their nonlabeled counterparts and are distinguishable by mass. Leucine enkephalin was included as a secondary reference compound and only 1 ion was monitored.

Figure A1

Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) spectrum of botulinum neurotoxin (BoNT) reactions. The top panel shows the MALDI-TOF-MS spectrum of the mixture of substrate peptides with no toxin present. The second panel shows this same mixture of substrate peptides and BoNT-A. The third, fourth and fifth panels show the mixture of substrate peptides with BoNT-B, -E, and –F, respectively. The insert mass spectrum for BoNT-E distinguishes the BoNT-dependent N-terminal product from the BoNT-A substrate peptide.