Electrospray ionization-tandem mass spectrometry analysis of the mycolic acid profiles for the identification of common clinical isolates of mycobacterial species

Mycolic acids are unique and complex molecular structures found in mycobacterial species. In the present study, we investigated whether electrospray ionization-tandem mass spectrometry (ESI-MS/MS) can be used to identify mycobacterial species based on their mycolic acid profiles. Clinical isolates of Mycobacterium tuberculosis complex and 18 nontuberculosis mycobacterial (NTM) species identified by PCR-restriction fragment length polymorphism (RFLP) or real-time PCR were used for this analysis. Crude lipid extracts were prepared by saponifying 1-2 colonies of individual isolates of mycobacterial species and by chloroform and methanol (2:1, v/v) extraction. ESI-MS/MS in negative ion mode with high cone voltage and collision energy was used for mycolic acid profiling analysis. Combinatorial precursor ion scans of m/z 395, 367, and 339 in the range of m/z 1000-1400 resulted in spectra specific to individual mycobacteria. M. tuberculosis complex and M. pulveris showed major ions by performing precursor ion scans on m/z 395 and 367, while other NTM species showed major ions by performing scans on m/z 367 and 339. The different NTM species examined showed different species dependent mycolic acid profiles. In conclusion, we describe a rapid, reliable, and informative ESI-MS/MS protocol for mycolic acid profiling in mycobacterial species, which allows mycobacterial species to be easily identified in clinical laboratories.