Sensitive profiling of chemically diverse bioactive lipids

Guanghou Shui, Anne K Bendt, Kevin Pethe, Thomas Dick, and Markus R Wenk

Department of Biochemistry and Department of Biological Sciences, National University of Singapore, Singapore, Singapore 117597

Corresponding Author: bchmrw{at}nus.edu.sg

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 (HPLC/ESI/QTOF-MS) experiment. The approach consists of: (i) sensitive isocratic elution which takes advantage of C18 column material that is resistant to elevated pH values induced by piperidine, (ii) chemometric alignment of mass spectra followed by differential analysis of ion intensities and (iii) semi-quantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts which harbor lipids of considerable chemical complexity. The method allows qualitative and semi-quantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides (PIMs), acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids. Mycolic acids are targets in antimycobacterial therapy and they play an important immunomodulatory role during host-pathogen interactions. We compared high resolution mass spectra of mycolic acids derived from Mycobacterium bovis BCG during entry into non-replicative conditions induced by oxygen deprivation (hypoxic dormancy). While the overall composition is not drastically altered there are pronounced differences in individual mycolic acids. Alpha-mycolic acids accumulate during entry into dormancy, while a sub-population of keto-mycolic acids 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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