Characterization of Mycolic Acids in Total Fatty Acid Methyl Ester Fractions from Mycobacterium Species by High Resolution MALDI-TOFMS

Abstract

Mycolic acids (MAs) are characteristic components of bacteria in the suborder Corynebacterineae, such as Mycobacterium. MAs are categorized into subclasses based on their functional bases (cyclopropane ring, methoxy, keto, and epoxy group). Since MAs have heterogeneity among bacterial species, analyzing of MAs are required in the chemotaxonomic field. However, their structural analysis is not easy because of their long carbon-chain lengths and several functional groups. In this study, total fatty acid (FA) methyl ester (ME) fraction of M. tuberculosis H37Rv was analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS). The distributions of carbon-chain length and their relative peak intensities were confirmed with those obtained by analysis of each subclass fraction which was separated from total FA ME fraction using thin-layer chromatography (TLC). The observed major peaks were reliably assigned as MAs owing to the high mass accuracy (error<3 ppm). The types of MA subclasses, their distributions of carbon-chain lengths, their relative peak intensities, and the ratio of even- and odd-numbered carbon-chain MAs for the total FA ME fraction were consistent with those of MA subclass fractions. To visualize whole MAs, contour maps of relative peak intensities for whole MAs were created. The contour maps indicated the MA subclasses and their distributions of carbon-chains with relative peak intensities at a glance. Our proposed method allows simple characterization in a short time and thus enables the analysis of large numbers of samples, and it would contribute to the chemotaxonomy.

Keywords: MALDI spiral-TOFMS; Mycobacterium; chemotaxonomy; contour map; mycolic acid.

Fig. 1. MA subclasses in M. tuberculosis.

TLC of MA ME (a). MALDI mass spectra of α-MA ME fraction (b), methoxy-MA ME fraction (c), and keto-MA ME fraction (d). RP indicates mass resolving power (FWHM). α, M, and K indicate α-, methoxy-, and keto-MAs, respectively.

Fig. 2. MALDI mass spectrum of the total FA ME fraction from M. tuberculosis.

α, M, and K indicate α-, methoxy-, and keto-MAs, respectively. Asterisk indicates the isotope peak corresponding to α-MA with C80.

Fig. 3. Comparisons of relative peak intensities of MA subclasses from M. tuberculosis.

α-MA (a), methoxy-MA (b), keto-MA (c), and corrected methoxy MA (d). Peak intensities were normalized such that the maximum peak intensity was 100%.

Fig. 4. MALDI mass spectra of total FA fractions of M. tuberculosis (a), M. bovis (b), M. kyorinense (c), and M. smegmatis (d).

Closed triangles (▲), inverted closed triangles (▼), open circles (○), closed squares (■), open squares (□), and closed circles (●) indicate α-, α′-, methoxy-, keto-, dicarboxy-, and epoxy-MAs, respectively.

Fig. 5. Contour maps of relative peak intensities for the whole MA contents of M. tuberculosis (a), M. bovis (b), M. kyorinense (c), and M. smegmatis (d).

Data are average peak intensities (a: n=5; b, c, and d: n=3). Size and color of each dot indicate peak intensity.