Top-Down Lipidomic Screens by Multivariate Analysis of High-Resolution Survey Mass Spectra

Schwudke, Dominik; Hannich, J. Thomas; Surendranath, Vineeth; Grimard, Vinciane; Moehring, Thomas J.; Burton, Lyle; Kurzchalia, Teymuras V.; Shevchenko, Andrej

doi:10.1021/ac062455y

Cited by 179 publications

(152 citation statements)

References 38 publications

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“…The most detailed analysis presented here required 100 min per sample. We note that the lipidomics platform can be operated with higher sample throughput as required for screening routines simply by lowering the structural specificity of the analysis (31). By monitoring the absolute abundance of individual lipid species we demonstrated that differences in growth temperature and defects in the lipid biosynthesis machinery produced alterations throughout the entire yeast lipidome.…”

Section: Discussionmentioning

confidence: 96%

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

Ejsing

Sampaio

Surendranath

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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963

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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 Ϸ95% 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.fatty acid elongation ͉ S. cerevisiae ͉ shotgun lipidomics T he lipidome of eukaryotic cells consists of hundreds to thousands of individual lipid species that constitute membranes, store metabolic energy and function as bioactive molecules (1-3). Despite the extensive characterization of proteins, their association into complexes and activities (4-6), it is still difficult to assess how perturbations within the lipid metabolic network affect the full lipidome of cells. This work shows that lipidome-wide quantification of individual molecular lipid species (molecules with defined chemical structure) by absolute quantification (expressed in mol or mol%) provides a new approach to relate lipidomics and functional genomics studies.The yeast Saccharomyces cerevisiae serves as a prime model organism for studying the molecular organization and regulatory circuitry of eukaryotic lipidomes (7-9). It uses a relatively simple and conserved network of lipid metabolic pathways (Fig. 1) that synthesize a few hundred molecular lipid species constituting its full lipidome (3). The lipidome diversity is primarily determined by the fatty acid synthase (10), the ⌬-9 desaturase (11) and the fatty acid elongation complex (12) that produce only saturated or mono-unsaturated fatty acids having 10 to 26 carbon atoms for the biosynthesis of glycerolipids, glycerophospholipids, and sphingolipids. Importantly, several metabolic conversions interlink sphingolipid, glycerophospholipid, and glycerolipid metabolism such that any perturbation within the metabolic network is prone to induce lipidome-wide ripple effects. Remarkably, numerous genes involved in lipid metabolism and trafficking can be mutated or deleted without apparent physiological consequences (Fig. 1).Despite remarkable methodological advances, lipidomics seldom complements functional genomics efforts owing to three major factors. First, analysis of glycerophospholipids and sphingolipids requires ...

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Section: Discussionmentioning

confidence: 96%

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

Ejsing

Sampaio

Surendranath

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

963

1,064

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“…Importantly, GSP profiling remains fully integrated into the shotgun lipidomics pipeline. High mass resolution of an LTQ-Orbitrap instrument enabled unequivocal assignment of species via their m/z , which was determined with a sub-ppm mass accuracy, hence eliminating the need of MS/MS (28,29). Therefore, it has become possible to profile the lipidome of GSPrich MDCK cells (19) and to track the lipidomic changes during epithelial polarization at the level of individual molecular species.…”

Section: Discussionmentioning

confidence: 99%

Membrane lipidome of an epithelial cell line

Sampaio

Gerl

Klose

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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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.epithelial polarity | shotgun lipidomics | high resolution mass spectrometry | Forssman glycosphingolipid | epithelial-mesenchymal transition G ain and loss of epithelial polarity are fundamental processes in tissue differentiation (1, 2). These processes have been extensively studied not only in the context of embryogenesis but in tumor progression and metastasis (1). Although cell polarization necessarily implies major changes in the structure and properties of the cell membranes, the exact changes in the molecular composition of their membrane lipidome remain unknown.Madin-Darby canine kidney (MDCK) cells are a cell culture model for epithelial polarization (3,4). When seeded at low density, MDCK cells undergo a morphological change from a fibroblast-like morphology to a well-polarized epithelium (5) and have recently also been adapted to undergo an epithelial-mesenchymal transition (EMT) (6). Epithelial cells have evolved characteristic apical and basolateral membrane domains, separated by tight junctions, with specific protein and lipid composition (7,8). The unusual robustness of the apical membrane is largely attributable to its special lipid composition (9-11).In this work, we applied shotgun MS for comprehensive and quantitative characterization of mammalian cellular membranes (12). By combining high mass resolution MS, an improved extraction procedure that increases the recovery of polar lipids (13), and the use of internal standard mixtures for absolute quantification, we expanded the lipid repertoire covered by shotgun lipidomics to glycosphingolipids (GSPs). Importantly, the analysis of the GSPs did not require the hydrolysis of glycerophospholipids (GPs) (14), and this simplification allowed us to determine the membrane lipidome with unprecedented comprehensiveness (more than 300 lipid species from 14 different lipid classes) from a single sample of 10 5 cells.We further elucidated how the lipidome is remodeled during the course of MDCK cell epithelial polarization. We found that when these c...

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“…In contrast, "top-down" lipidomic approaches in which all structure elucidation occurs on ionized and mass-selected lipids inside the mass spectrometer have rapid throughput and limited sample requirements. However, a current limitation of such approaches is that key structural motifs that may play critical roles in defining the specificity of lipid-protein interactions [19,20] remain unresolved or ambiguous. More specifically, "top-down" techniques cannot readily determine: (1) the position of double bonds within an acyl chain; (2) the stereochemistry of double bond(s) (i.e., cis or trans); and (3) the relative position of acyl chains on the backbone of a complex lipid (i.e., sn-position on a glycerolipid).…”

Section: Krylova Et Al Observed An [M -H]mentioning

confidence: 99%

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

Brown

Mitchell

Oakley

et al. 2012

J. Am. Soc. Mass Spectrom.

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Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.Key words: Lipid-protein interactions, Structural biology, Structural lipidomics, Protein mass spectrometry Lipid-Protein Interactions P roteins and lipids, both integral for the function of all biological systems, are chemically distinct. A protein's three-dimensional structure and ultimately its function are defined by peptide-bonded amino acids and subsequent posttranslational modifications. In contrast, lipids are smaller molecules than proteins, but have a larger array of building blocks and linkage chemistries. Because of these inherent chemical differences, the optimal tools for de novo structural characterization differ between proteins and lipids.Lipid-protein interactions are critical for maintaining biological function in the membrane bilayer, the cytosol, and extracellular space. In the membrane, the chemical structures of lipid solvent molecules are important for the correct folding, insertion, structure, and function of membrane proteins. The interactions between enzymes and their respective lipid substrates are critical for correct substrate recognition and catalysis (e.g., lipoxygenase [1]). Additionally, lipid-protein interactions are increasingly being realized as important in maintaining cellular structure (e.g., cytoskeletal anchoring [2]), scaffolding and localization of multi-subunit protein complexes (e.g., associated kinase anchoring protein complexes [3]), and as second messengers in signal transduction (e.g., protein kinase C [4]).

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Top-Down Lipidomic Screens by Multivariate Analysis of High-Resolution Survey Mass Spectra

Cited by 179 publications

References 38 publications

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

Membrane lipidome of an epithelial cell line

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

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