Identification of microbial mixtures by capillary electrophoresis/selective tandem mass spectrometry

In this paper, we propose a new strategy for identifying specific bacteria in bacterial mixtures by using CE-selective MS/MS of peptide marker ions associated with the bacteria of interest. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that match the protein associated with the corresponding species were selected as marker ions for bacterial identification. Specific peptide marker ions were obtained for each of the following three pathogens: Pseudomonas aeruginasa, Staphylococcus aureus, and Staphylococcus epidermidis. To identify a bacterial species in a sample, we performed CE-MS/MS analysis of the selected marker ions in the proteolytic digest of the cell extract and then performed protein database searches. The selected peptides that we identified correctly from Xcorr values ranking at the top of the search results allowed us to identify the corresponding bacterial species present in the sample. We have applied this method successfully to the identification of various mixtures of the three pathogens. Even minor bacterial species present at a concentration of 1% can be identified with great confidence. This method for CE-MS/MS analysis of bacteria-specific marker peptides provides excelent selectivity and high accuracy when identifying bacterial species in complex systems. In addition, we have used this approach to identify P. aeruginasa in a saliva sample spiked with E. coli and P. aeruginasa.