Vitali Matyash, Gerhard Liebisch, Teymuras V. Kurzchalia, Andrej Shevchenko, Dominik Schwudke

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided ≈95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.

Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin–Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet.

Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

LipidXplorer is the open source software that supports the quantitative characterization of complex lipidomes by interpreting large datasets of shotgun mass spectra. LipidXplorer processes spectra acquired on any type of tandem mass spectrometers; it identifies and quantifies molecular species of any ionizable lipid class by considering any known or assumed molecular fragmentation pathway independently of any resource of reference mass spectra. It also supports any shotgun profiling routine, from high throughput top-down screening for molecular diagnostic and biomarker discovery to the targeted absolute quantification of low abundant lipid species. Full documentation on installation and operation of LipidXplorer, including tutorial, collection of spectra interpretation scripts, FAQ and user forum are available through the wiki site at: https://wiki.mpi-cbg.de/wiki/lipidx/index.php/Main_Page.