Evolution of Mass Spectrometry in Laboratory Testing of Biothreat Agents

Introduction: Clinical, agricultural and public health laboratories (PHL) screen thousands of samples daily for bacterial agents and toxins. Sample preparation, processing and analysis using conventional microbiological methods can range from days to weeks for pathogen identification. Two agents of high concern are Botulinum neurotoxin (BoNT) which cause the disease known as botulism by inhibiting neurotransmitter release at the neuromuscular junction and ricin toxin which inhibits protein synthesis

Purpose: Both methods require extensive and cumbersome processing leading to time consuming testing as negative results can take up to four days to identify. As the number of specimens has increased in laboratories, there exists a great need to quickly identify sources of exposure which has led our laboratory to explore the use of Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS).

Methods: An endopeptidase-based mass spectrometry method, which was developed by the CDC, has been transitioned to be used with the Bruker Daltonics MALDI-TOF Biotyper. This MS method utilizes the endoproteinase activity of the toxin to identify all BoNT types (A-G) with a MALDI-TOF/MS by cleaving peptides at specific sites.  Each toxin is identified by the mass-to-charge ratios of these fragmented peptides for BoNT and the depurination of a RNA substrate for ricin toxin.

Results: For each assay, BoNT and ricin toxins were spiked into over ten different food matrices to assess detection capability. Assay sensitivity for BoNT types A, B, E, and F ranged from 0.3 to 25 MLD_50­.  Active ricin toxin was detected as low as 5 mg/ml.

Significance: Our findings support identification of BoNT and ricin toxin using the mass spectrometry-based method, replacing the need for performing traditional costly, time-consuming methods. Furthermore, this method eliminates the need to use animals for laboratory testing.