Targeted proteomics approach to species-level identification of Bacillus thuringiensis spores by AP-MALDI-MS.
Anthrax infections progress at a rapid pace, making rapid detection methods of utmost importance. MALDI-MS proteomics methods focused on Bacillus anthracis detection have targeted chromosomally encoded proteins, which are highly conserved between closely related species, hindering species identification. Presented here is an AP-MALDI-MS method targeting plasmid-borne proteins from Bacillus spores for species-level identification. A bioinformatics analysis revealed that 60.3% and 75.4% of tryptic peptides from plasmid-borne proteins of B. anthracis and B. thuringiensis were species-specific, respectively. Reported here is a method in which plasmid-borne delta-endotoxins were extracted directly from B. thuringiensis spores in 100 mM KOH. The pH was then adjusted to 8 and a 5-min trypsin digestion was performed on the extracted proteins. The resulting tryptic peptides were analyzed by AP-MALDI-MS/MS, which produced a definitive identification the B. thuringiensis species-specific Cry1Ab protein with a MASCOT score of 278 and expect value of 7.5 x 10(-23). This method has demonstrated the detection and identification of B. thuringiensis spores at the species level following a 5-min trypsin digestion. The challenges in applying a similar approach to the detection of plasmid-borne protein toxins from B. anthracis are also discussed.
Department of Chemistry, California State University, Stanislaus, Turlock, California 95382-0299, USA.
Jennifer
J
Nguyen
Scott C
SC
Russell
D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000145:classification*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000302:isolation & purification; D006460:Hemolysin Proteins / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013171:Spores, Bacterial / Q000737:chemistry / Q000145:classification; D014357:Trypsin / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014357:Trypsin