Mass spectrometric identification and differentiation of botulinum neurotoxins through toxin proteomics

Abstract

Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence and immunogenic properties, and some subtypes are further differentiated into toxin variants. Toxin characterization is important as different types of BoNT can respond differently to medical countermeasures for botulism, and characterization of the toxin can aid in epidemiologic and forensic investigations. Proteomic techniques have been established to determine the serotype, subtype, or toxin variant of BoNT. These techniques involve digestion of the toxin into peptides, tandem mass spectrometric (MS/MS) analysis of the peptides, and database searching to identify the BoNT protein. These techniques demonstrate the capability to detect BoNT and its neurotoxin-associated proteins, and differentiate the toxin from other toxins which are up to 99.9% identical in some cases. This differentiation can be accomplished from toxins present in a complex matrix such as stool, food, or bacterial cultures and no DNA is required.

Rapid and sensitive detection of botulinum toxin type A in complex sample matrices by AlphaLISA

“…As a potential diagnostic antibody for BoNT/A, it has good specificity and does not exhibit cross reaction with enterotoxins and BoNT/B. It could clearly distinguish between BoNT/A and BoNT/B, which is very important for botulism detection, because different serotypes of botulism need to be neutralized with specific antisera ( 16 , 17 ).…”

“…For antigen-antibody reactions, it has previously been reported that the acidity of freshly squeezed orange juice and the fat or casein in milk can affect antigen-antibody binding, which could negatively affect the assay ( 15 ). To reduce the effects of the food matrix, some studies isolated, tested, and quantified toxins from food samples ( 3 , 16 ). In this study, the AlphaLISA method was not only found to be sensitive and rapid, but also showed a certain tolerance to a variety of different sample type matrices.…”

“…However, compared with orange juice, the pH of gastric juices 0.9–1.5, much lower than that of orange juice. Because BoNT substrate neurotoxin-associated proteins (NAPs) can protect the toxin from digestion when exposed to pepsin at pH 2, the matrix proteins are digested and the toxin complex remains intact ( 16 ), which is also one reason why the toxin can be detected in vomit by mass spectrometry. Here, however, since we did not try a lower pH than orange juice, we did not determine the detection ability of AlphaLISA for lower pH samples.…”

Biological toxins of potential bioterrorism risk: Current status of detection and identification technology

“…An arsenal of different methods has been developed in the BoNT field which can be roughly grouped into (i) immunological assays detecting the presence of the molecule [34], (ii) in vitro biochemical methods such as Endopep-ELISA and Endopep-MS detecting the catalytic activity and the serotype [35,36], and (iii) the functional in vitro cell-based assay [37], ex vivo mice phrenic nerve (MPN) hemidiaphragm assay [38], and in vivo mouse bioassay (MBA) [34] which measure the full biological activity and toxicity. However, apart from the mouse bioassay [39] and one sandwich ELISA [40], none of the other methods had been evaluated in a PT or method performance study until recently.…”

Proteomics Goes to Court: A Statistical Foundation for Forensic Toxin/Organism Identification Using Bottom-Up Proteomics

“…For example, proteomics has been used to characterize items of historical and archeological significance, including ancient paints, , tuberculosis-ridden skeletal remains, and South American ungulates from the last Ice Age . A number of law enforcement applications have been described, including microbial identification, − characterization of microbiological growth media, human identification from hair samples, , identification of bodily fluids, − species identification using bones − and other tissues, as well as detection of protein toxins such ricin, − the botulinum neurotoxin, , and others. ,, …”