A novel MALDI-TOF mass spectrometry sample preparation strategy for improved proteomic biotyping of clinically significant Mycobacteria

A precise routine method is essential for the rapid identification of pathogenic mycobacterial species to support our publicly maintained health care management systems to efficiently treat and control and emerging tuberculosis pandemic that is threatening populations of global third world economies. To date, many conventional and more recently developed molecular genotyping methods are employed to this end. However, current technologies are limited in respect in that they are time consuming nature and dependent on highly skilled technical personnel expertise. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently reported as reliable, economical, and highly efficient method for both bacteria, yeast and to a limited extent for mycobacterial strain identification. Unlike other microbes, a major impediment exists in the efficient extraction of cellular proteins from mycobacteria, especially due to their complex cell wall associated lipid structure. In this study, the manufacturer prescribed mycobacterial sample preparation method for MALDI TOF MS is modified and optimised to generate more efficient cellular protein extraction for efficient biotyping. In this regard, the newly developed sample preparation method is inclusive of a glass bead cellular disruption/delipidation followed by a chloroform-methanol solvent extraction event. Interestingly, the data from this locally based study shows that newly developed method generates unique and highly reproducible mass spectra profiles. A new and independent main spectral profile reference library (CMEFA-MSP) representing clinically relevant American Type Culture Collection (ATCC) mycobacterial strains and clinical isolates was established and subsequently used to unequivocally identify 110 (n = 100) blind-coded clinical mycobacterial isolates to the species level that displayed log score values of ≥2.3. This strongly suggests that MALDI-TOF MS when used in conjunction with the CMEFA sample preparation protocol has potential as a simple and cost-effective alternative for the unambiguous identification of clinically important mycobacteria.