Proteomic Profiling of Intact Mycobacteria by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

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Abstract
Current methods for the identification of mycobacteria in culture are time-consuming, requiring as long as 12 weeks for positive identification. One potential approach to rapid mycobacterial identification is to utilize proteomic profiling of cultures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this report, we have applied MALDI-TOF MS to proteomic profiling of cultured microorganisms representing six species of the genus Mycobacterium. We find that analysis of acetonitrile/trifluoroacetic acid cellular extracts produces data similar to that of the analysis of deposited whole cells, while minimizing human contact with the microorganisms and rendering them nonviable. A matrix composition of α-cyano-4-hydroxycinnamic acid with fructose yields highly reproducible MALDI-TOF spectra. Statistical analysis of MALDI-TOF MS data allows differentiation of each individual mycobacterial species on the basis of unique mass fingerprints. The methodology allows identification of a number of unique (potentially diagnostic) biomarkers as targets for protein identification by MS/MS experiments. In addition, we observe a number of signals common to all mycobacterial species studied by MALDI-TOF MS, which may be genus-specific biomarkers. The potentially genus-specific biomarkers occur at low mass (<2 kDa) and are likely to be lipids and cell wall components such as mycolic acids. This study demonstrates the potential for mass spectrometry-based identification/classification of mycobacteria.