出版社：American Society for Biochemistry and Molecular Biology
摘要：Here, we present an improved method for sensitive profiling of lipids in a single high-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry experiment. The approach consists of i ) sensitive isocratic elution, which takes advantage of C18 column material that is resistant to increased pH values induced by piperidine, ii ) chemometric alignment of mass spectra followed by differential analysis of ion intensities, and iii ) semiquantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts that harbor lipids of considerable chemical complexity. The method allows qualitative and semiquantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, and glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides, acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids (MAs). MAs are targets in antimycobacterial therapy, and they play an important immunomodulatory role during host-pathogen interactions. We compared high-resolution mass spectra of MAs derived from Mycobacterium bovis Bacille Camette-Guérin during entry into nonreplicative conditions induced by oxygen deprivation (hypoxic dormancy). Although the overall composition is not drastically altered, there are pronounced differences in individual MAs. α-MAs accumulate during entry into dormancy, whereas a subpopulation of keto-MAs is almost entirely eliminated. This effect is reversed upon resuscitation of dormant mycobacteria. These results provide detailed chemical information with relevance to drug development and immunobiology of mycobacteria.
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