Identification of mycolic acid class ratios from Mycobacterium species using liquid chromatography-mass spectrometry

Abstract

Alongside the recent technology advancements in liquid chromatography (LC) and mass spectrometry (MS) instrumentation there has also been steady progress made with respect to the systematic lipidomic studies of Mycobacterium tuberculosis (M. tb) where hyphenated LC and MS instrumentation were the main analytical technique employed. Although many reports have shown low limits of detection and high precision in analysing mycolic acids (MAs) and other M. tb lipids, few have reported efficient LC selectivity, even when employing high resolution mass spectrometry (HRMS) to determine the accurate mass of a lipid molecule for identification purposes. MAs are one of the most widely reported lipid classes that are found in the cell wall of M. tb and consist of a diversity of high mass branched chain of fatty acids that could potentially be used as diagnostic biomarkers for determining active M. tb infections. The chemical diversity of MAs can be used for taxonomic identification of mycobacterial species, but their analysis is complicated due to their extreme hydrophobic properties and homologous chemistry. Three dominant subclasses of MA molecules are found in mycobacteria: alpha-(α-), ketoand methoxy-MAs. When analysing MAs using MS, the product ions obtained from the infusion of a purified mixture of MAs, extracted from M. tb H37Rv strain, on a tandem mass spectrometer (MS/MS) are m/z 367.3 and 395.4, which correlate with published data but are not unique to single MA classes and therefore cannot be used for the identification of a specific MA subclass molecule. The ratios of the diverse MA class precursor ions can however be used to predict the identity of M. tb strains. Further genetic assays are however required to confirm the taxonomic identification. In this study, self-extracted MAs (M. tb H37Rv strain) were compared to that of commercially available MAs (M. bovis strain) and by integrating the chromatographic peak area of each dominant precursor ion within a MA class, a peak area ratio between the α-, keto- and methoxy- MAs were determined. The resulting percentage ratios between α-, keto- and methoxy- MA classes were found to be 53:8:38 for M. tb H37Rv and 53:15:32 for M. bovis respectively. The LC data dependent acquisition (DDA) precursor ion method, developed here specifically for the MA ratio comparison, has also shown the ability to resolve isomers within a MA class that has not previously been reported. Two other classes of mycobacterial lipids, phthiocerol dimycocerosates (DIMa) and phthiodiolone dimycocerosates (DIMb), collectively called PDIMs, have recently emerged as lipids which play a significant role in the virulence of drug resistant M. tb. Hence, a robust LC-HRMS method was developed for the analysis of extractable non-polar lipids extracted from three different drug resistant phenotypes of M. tb from clinical isolates and compared to a drug susceptible M. tb H37Rv clinical isolate. dimycocerosates (DIMa) and phthiodiolone dimycocerosates (DIMb), collectively called PDIMs, have recently emerged as lipids which play a significant role in the virulence of drug resistant M. tb. Hence, a robust LC-HRMS method was developed for the analysis of extractable non-polar lipids extracted from three different drug resistant phenotypes of M. tb from clinical isolates and compared to a drug susceptible M. tb H37Rv clinical isolate. In future, the feasibility of using LC-HRMS as a routine technique to phenotype M. tb drug resistant strains may require a mass analyser with a mass resolution in excess of 100 000, as well as chromatographic technologies that are capable of resolving non-derivatized complex mixtures of large (C60-C100) nonpolar and/or polar lipid molecules.