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Tandem Mass Spec for Select Agent Detection

Efforts to mitigate biothreats require development of efficient countermeasures which in turn relies on fast and accurate methods to detect the biological agents in a range of complex matrices including environmental and clinical samples.

Recent developments in the mass spectrometry (MS) technology promise unequivocal identification of microorganisms with remarkable specificity, speed, and sensitivity. The multiplexing capability and suitability for analyzing complex samples such as air, water, culture medium, bodily fluids, and food are of immense value for the utility of MS based techniques in biothreat scenario.

Researchers in this paper report a comprehensive elucidation of marker proteins and species specific unique peptides for the targeted tandem MS analysis of bacterial select agents. The targeted approach described is distinct from the well-known multiple reaction monitoring (MRM) assays and involves selection of several unique peptide masses for the tandem MS analysis after the first MS scan.

A combination of shot-gun MS/MS analysis and the targeted search using a panel of unique peptides is likely to provide unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples. The comprehensive list of peptides reflected in the inclusion list, makes a valuable resource for the multiplex analysis of select biothreat agents and further development of targeted MS/MS assays.

The species-specific marker peptides for the nine bacterial select agents were elucidated by extensive literature mining, in silico analysis, and MS/MS analysis of abundant proteins from axenically grown bacterial species in our laboratory. As a proof of concept, the methodology was validated using blind samples spiked with one or two select agents spiked in soil and sand.

Researchers leading this study are affiliated with the Biotechnology Division, Defence Research & Development Establishment, Gwalior, India; and the Microbiology Division, Defence Research & Development Establishment, Gwalior, India.

Rajoria, S., Sabna, S., Babele, P. et al. Elucidation of protein biomarkers for verification of selected biological warfare agents using tandem mass spectrometry. Scientific Reports. Published: 10 February 2020 https://doi.org/10.1038/s41598-020-59156-3


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