Multiplex Detection of Protein Toxins Using MALDI-TOF-TOF Tandem Mass Spectrometry: Application in Unambiguous Toxin Detection from Bioaerosol

Abstract

Protein toxins, such as botulinum neurotoxins (BoNTs), Clostridium perfringens epsilon toxin (ETX), staphylococcal enterotoxin B (SEB), shiga toxin (STX), and plant toxin ricin, are involved in a number of diseases and are considered as potential agents for bioterrorism and warfare. From a bioterrorism and warfare perspective, these agents are likely to cause maximum damage to a civilian or military population through an inhalational route of exposure and aerosol is considered the envisaged mode of delivery. Unambiguous detection of toxin from aerosol is of paramount importance, both for bringing mitigation protocols into operation and for implementation of effective medical countermeasures, in case a "biological cloud" is seen over a population. A multiplex, unambiguous, and qualitative detection of protein toxins is reported here using tandem mass spectrometry with MALDI-TOF-TOF. The methodology involving simple sample processing steps was demonstrated to identify toxins (ETX, Clostridium perfringes phospholipase C, and SEB) from blind spiked samples. The novel directed search approach using a list of unique peptides was used to identify toxins from a complex protein mixture. The bioinformatic analysis of seven protein toxins for elucidation of unique peptides with conservation status across all known sequences provides a high confidence for detecting toxins originating from any geographical location and source organism. Use of tandem MS data with peptide sequence information increases the specificity of the method. A prototype for generation of aerosol using a nebulizer and collection using a cyclone collector was used to provide a proof of concept for unambiguous detection of toxin from aerosol using precursor directed tandem mass spectrometry combined with protein database searching. ETX prototoxin could be detected from aerosol at 0.2 ppb concentration in aerosol.

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

“…This indicates the need to develop simple but more effective sample preparation procedures for MS-based methods with a high confirmative power to eliminate as many interfering substances as possible before the detection step. In addition and in parallel to the important issue of sample preparation, the development of stand-alone or hyphenated mass spectrometry methods, which should offer more reliable toxin identification and, in the medium term, accurate quantification of biological toxins, will gain increased attention [74,75]. Additionally, MS-based methods offer the technical advantage of providing detailed information on toxin contents, both known and unknown, especially when using scanningmode MS approaches, thereby adding an open view to the diagnostic or forensic workflow [76].…”

Proteome-based bacterial identification using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS): A revolutionary shift in clinical diagnostic microbiology

“…Alam et al described a multiplex and qualitative detection of protein toxins using MALDI-TOF-TOF [85]. The methodology was useful to identify toxins including C. perfringens epsilon toxin and staphylococcal enterotoxin.…”

Biological Toxins and Bioterrorism

“…The toxin is composed of a 27 kDa monomer A subunit linked to the B domain by a trypsin-sensitive loop and a long a-helix, which inserts Alam et al (2012) inside the core of the B pentamer, thus anchoring the two subunits. It also has an activated A1 chain, disulfide linked to an A2 chain that remains tightly associated with a pentameric ring of B subunits.…”