Background: Some pathogenic bacteria can be potentially used for nefarious applications
in the event of bioterrorism or biowarfare. Accurate identification of biological agent from
clinical and diverse environmental matrices is of paramount importance for implementation of medical
countermeasures and biothreat mitigation.
Objective: A novel methodology is reported here for the development of a novel enrichment strategy
for the generally conserved abundant bacterial proteins for an accurate downstream species identification
using tandem MS analysis in biothreat scenario.
Methods: Conserved regions in the common bacterial protein markers were analyzed using bioinformatic
tools and stitched for a possible generic immuno-capture for an intended downstream
MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs
were generated for the expression of conserved stitched regions of 60 kDa chaperonin
GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein.
Results: The conserved regions of common bacterial proteins were stitched for a possible generic
immuno-capture and subsequent specific identification by tandem MS using variable regions of the
molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were
generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study,
hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with
~60 KDa proteins from the cell lysates of three bacterial species tested.
Conclusion: The envisaged methodology can lead to the development of a novel enrichment strategy
for the abundant bacterial proteins from complex environmental matrices for the downstream
species identification with increased sensitivity and substantially reduce the time-to-result.