Abstract
Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.
Highlights
Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism
Many lipidomic methods that involve the direct measurement of lipid species from lipid extracts are based on direct infusion into ESI sources or ionization by MALDI [16, 17], in which the detection of less abundant molecular species is typically impeded by ion suppression
The use of a 10 mM ammonium acetate buffer adjusted with formic acid to pH 5.5, a pH at which all major phospholipids have an equable charge state [34, 35], gave an acceptable separation of the lipid extract of M. xanthus DK1622 [36] as depicted in Fig. 2, while ESI-MS/MS analysis allowed the detection and identification of eluting lipid species
Summary
Cells were harvested by centrifugation at 15,000 g for 15 min at room temperature and washed once with the same volume of distilled H2O. All chromatographic traces were smoothed using a Gaussian algorithm (1 pt width, 2 cycles) to enhance peak detection Those EICs from the identified lipid species were integrated by applying the “Compounds Ϫ Chromatograms” algorithm with the following settings: signal-tonoise threshold, 5; area threshold, 10%; intensity threshold, 10,000 for the negative ion mode and 1,000,000 for the positive ion mode; skim ratio, 0; smoothing width, 1. High resolution MS analyses Determination of the exact mass of Cer molecular species from lipid extracts was carried out using a Dionex Ultimate 3000 RSLC coupled to a Bruker micrOTOF-Q II equipped with an ESI source set to negative ionization mode. Nomenclature For the sake of uniformity and comprehensibility, we used the nomenclature of the LIPID MAPS Lipid Classification System [24] for the names and abbreviations
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