Quantification of Botulinum Neurotoxin Serotypes A and B from Serum Using Mass Spectrometry

Botulinum neurotoxins (BoNT) are the deadliest agents known. Previously, we reported an endopeptidase activity based method (Endopep-MS) that detects and differentiates BoNT serotypes A–G. This method uses serotype specific monoclonal antibodies and the specific enzymatic activity of BoNT against peptide substrates which mimic the toxin’s natural target. Cleavage products from the reaction are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We have now developed a multiple reaction monitoring method to quantify the biological activity of BoNT serotypes A (BoNT/A) and B (BoNT/B) present in 0.5 mL of serum using electrospray mass spectrometry. The limit of quantification for each serotype is 1 mouse intraperitoneal lethal dose (MIPLD₅₀) corresponding to 31 pg of BoNT/A and 15 pg of BoNT/B in this study. This method was applied to serum from rhesus macaques with inhalational botulism following exposure to BoNT/B, showing a maximum activity of 6.0 MIPLD₅₀/mL in surviving animals and 653.6 MIPLD₅₀/mL in animals that died in the study. The method detects BoNT/B in serum 2–5 h after exposure and up to 14 days. This is the first report of a quantitative method with sufficient sensitivity, selectivity, and low sample size requirements to measure circulating BoNT activity at multiple times during the course of botulism

Amino acid sequence of peptides used in and produced by the Endopep-MS method.

<p>The observed <i>m/z</i> of each peptide is also included.</p

Sequence alignment of known BoNT subtypes in the range of Y750 to E757 of BoNT/A.

<p>Residues that play an important role in 4E17.1 binding are bolded and underlined. Dissociation rates (K_D) in pM of BoNT with mAb 4E17.1 are also listed. NB indicates no binding was observed and ND indicates that the K_D was not determined. Each BoNT is also identified by strain tested where appropriate. Equilibrium dissociation constant (K_D) were measured by flow fluorimetry in a KinExA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012237#pone.0012237-GarciaRodriguez1" target="_blank">[17]</a>.</p

Mass spectra of the Endopep-MS BoNT/F reactions with /F1 (4A), /F6 (4B), /F2 (4C), or no toxin (4D) extracted using the 4E17.1 antibody-coated beads.

<p>The peptide cleavage products indicating BoNT/F are <i>m/z</i> 1345.2 and 3167.8 and the peptide substrate is present at <i>m/z</i> 4497.5.</p

Mass spectra of the Endopep-MS BoNT/A reactions with /A1 (1A), /A2 (1B), /A3 (1C), /A4 (1D), or no toxin (1E) extracted using the 4E17.1 antibody-coated beads.

<p>The peptide cleavage products indicating BoNT/A are <i>m/z</i> 1197.8 and 1699.9; the peptide substrate is present at <i>m/z</i> 2878.7.</p

Mass spectra of the Endopep-MS BoNT/E reactions with /E1 (3A), /E2 (3B), /E3 (3C), /E4 (3D), or no toxin (3E) extracted using the 4E17.1 antibody-coated beads.

<p>The peptide cleavage products indicating BoNT/E are <i>m/z</i> 1136.6 and 2923.6 and the peptide substrate is present at <i>m/z</i> 4041.1.</p

Mass spectrum of the Endopep-MS reaction of the simultaneous extraction of BoNT/A, /B, /E, and /F with monoclonal antibody 4E17.1 (6A) or the single extraction of BoNT/A (6B), /B(6C), /E (6D), or /F (6E) with serotype-specific antibodies.

<p>Cleavage products indicating those four BoNTs are marked.</p

Mass spectra of the Endopep-MS BoNT/D reactions with no antibody extraction (5A), extraction with monoclonal antibody 4E17.1 (5B), or extraction with monoclonal antibody CR2 (5C).

<p>The peptide cleavage products indicating BoNT/D are <i>m/z</i> 1217.7 and 3296.9 and the peptide substrate is present at <i>m/z</i> 4497.5.</p

Mass spectra from the reaction of peptide substrates with 4E17.1 coated beads used to extract A2b (7A), Ba4 (7B), Af (7C), and Bf (7D).

<p>Peaks at <i>m/z</i> 1197.8 and 1699.9 indicate BoNT/A, peaks at <i>m/z</i> 1759.9 and 2283.4 indicate BoNT/B, and peaks at <i>m/z</i> 1345.2 and 3167.8 result from BoNT/F activity.</p

Mass spectra of the Endopep-MS BoNT/B reactions with /B1 (2A), /B2 (2B), /B3 (2C), /B4 (2D), /B5 (2E), or no toxin (2F) extracted using the 4E17.1 antibody-coated beads.

<p>The peptide cleavage products indicating BoNT/B are <i>m/z</i> 1759.9 and 2283.4 and the peptide substrate is present at <i>m/z</i> 4024.7.</p