Accurate molecular mass determination of mycolic acids by MALDI-TOF mass spectrometry

Mycolic acids, major and specific long-chain fatty (C70-C90) acid component s of the mycobacterial cell envelope, were analyzed for the first time usin g matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) ma ss spectrometry operating in a reflectron mode. The various types of purifi ed mycolates from representative mycobacterial species were analyzed using 2,5-DHB as matrix, because less than 10 pmol of mycolates was sufficient to obtain well-resolved mass spectra composed exclusively of pseudomolecular [M + Na](+) ions consistent with the structures deduced from the chemical a nalytical techniques applied to these molecules. Examination of the MALDI m ass spectra demonstrated that the chain lengths of the various mycolates co rrelated with the growth rate of mycobacterial strains. Although slow growe rs, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, produced a series of odd carbon numbers (C74-C82) of alpha -mycolic acids, rapid gr owers synthesized both odd and even carbon numbers. In addition, the main c hain of oxygenated mycolic acids from slow growers were four to six carbon atoms longer than the corresponding alpha -mycolic acids, whereas rapid gro wers elaborated oxygenated homologues possessing the same chain lengths as their alpha -mycolic acids. Furthermore, a comparative analysis of the crud e fatty acid mixtures from a wild-type strain of M. tuberculosis and its is ogenic mutant effected in the synthesis of oxygenated mycolates by MALDI ma ss spectrometry revealed structural differences between the alpha -mycolate s from the two strains. Thus, this technique appeared to be a rapid and hig hly sensitive technique for the analysis of mycolic acids, not only by prov iding accurate molecular masses and new structural information, but also by both reducing sample consumption and saving time.