Characterization of botulinum neurotoxin type A subtypes by immunocapture enrichment and liquid chromatography–tandem mass spectrometry

Botulinum neurotoxins (BoNT) are divided into seven toxinotypes based on their immunological properties and each toxinotype contains several subtypes according to their amino acid sequences. Here, we designed a mass spectrometry method able to identify BoNT/A subtypes in complex matrices including crude culture supernatants, food, and environmental samples. Peptides from BoNT light chain (L) specific to the subtypes BoNT/A1 to A3 and BoNT/A5 to A8 were identified. The method consists of an immunocapture step with antibodies specific to BoNT/A L chains followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) on a triple quadrupole mass spectrometer (QqQ) in multiple reaction monitoring (MRM) mode. BoNT/A subtypes were correctly identified in culture supernatants and in tap water or orange juice samples with a limit of detection of 20 to 150 mouse lethal doses (MLD) and with a lower sensitivity in serum samples.