Abstract
Some pathogens and toxins have the potential to be used as weapons of mass destruction and instigate population-based fear. Efforts to mitigate biothreat 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. We report here an mass spectrometry (MS) based methodology, employing both targeted and shot-gun approaches for the verification of biological agents from the environmental samples. Our shot-gun methodology relied on tandem MS analysis of abundant peptides from the spiked samples, whereas, the targeted method was based on an extensive elucidation of marker proteins and unique peptides resulting in the generation of an inclusion list of masses reflecting relevant peptides for the unambiguous identification of nine bacterial species [listed as priority agents of bioterrorism by Centre for Disease Control and Prevention (CDC)] belonging to phylogenetically diverse genera. The marker peptides were elucidated by extensive literature mining, in silico analysis, and tandem MS (MS/MS) analysis of abundant proteins of the cultivated bacterial species in our laboratory. 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.
Highlights
Some pathogens and toxins have the potential to be used as weapons of mass destruction and instigate population-based fear
The realization that that some pathogenic microbes and toxins of biological origin can be used as weapons of mass destruction has gained prominence especially in the backdrop of growing concerns for bioterrorism
Our shot-gun methodology relied on tandem mass spectrometry (MS) analysis of abundant peptides from the spiked samples, whereas, the targeted method was based on an extensive elucidation of marker proteins, unique peptides, strain coverage analysis, and generation of an inclusion list of masses reflecting relevant peptides for the unambiguous identification of selected bacterial biowarfare agents
Summary
Some pathogens and toxins have the potential to be used as weapons of mass destruction and instigate population-based fear. Our shot-gun methodology relied on tandem MS analysis of abundant peptides from the spiked samples, whereas, the targeted method was based on an extensive elucidation of marker proteins and unique peptides resulting in the generation of an inclusion list of masses reflecting relevant peptides for the unambiguous identification of nine bacterial species [listed as priority agents of bioterrorism by Centre for Disease Control and Prevention (CDC)] belonging to phylogenetically diverse genera. MALDI Biotyper and VITEK MS have been approved by the U.S Food and Drug Administration for bacterial identification in clinical diagnostic laboratories[23,24,25,26] Both these MALDI-TOF-MS-based platforms ionize intact proteins extracted from whole cell culture without specific protease treatment[19,27]. Mesuere et al.[43] have recently reported a web application, The Unique Peptide Finder (http://unipept.ugent.be/peptidefinder), to screen for taxon-specific tryptic peptides
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