Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry

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Abstract
Direct estimation of the molecular mass of single molecular species of trehalose 6-monomycolate (TMM), a ubiquitous cell-wall component of mycobacteria, was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. When less than 1 μg TMM was analysed by MALDI-TOF mass spectrometry, quasimolecular ions [M+Na]+of each molecular species were demonstrated and the numbers of carbons and double bonds (or cyclopropane rings) were determined. Since the introduction of oxygen atoms such as carbonyl, methoxy and ester groups yielded the appropriate shift of mass ions, the major subclasses of mycolic acid (α, methoxy, keto and wax ester) were identified without resorting to hydrolytic procedures. The results showed a marked difference in the molecular species composition of TMM among mycobacterial species. Unexpectedly, differing from other mycoloyl glycolipids, TMM fromMycobacterium tuberculosisshowed a distinctive mass pattern, with abundant odd-carbon-numbered monocyclopropanoic (or monoenoic)α-mycolates besides dicyclopropanoic mycolate, ranging from C75to C85, odd- and even-carbon-numbered methoxymycolates ranging from C83to C94and even- and odd-carbon-numbered ketomycolates ranging from C83to C90. In contrast, TMM fromMycobacterium bovis(wild strain and BCG substrains) possessed even-carbon-numbered dicyclopropanoicα-mycolates. BCG Connaught strain lacked methoxymycolates almost completely. These results were confirmed by MALDI-TOF mass analysis of mycolic acid methyl esters liberated by alkaline hydrolysis and methylation of the original TMM. Wax ester-mycoloyl TMM molecular species were demonstrated for the first time as an intact form in theMycobacterium avium–intracellularegroup,M. phleiandM. flavescens. TheM. avium–intracellularegroup possessed predominantly C85and C87wax ester-mycoloyl TMM, whileM. phleiand the rapid growers tested contained C80, C81, C82and C83wax ester-mycoloyl TMM. This technique has marked advantages in the rapid analysis of not only intact glycolipid TMM, but also the mycolic acid composition of each mycobacterial species, since it does not require any degradation process.

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