LipidXplorer: A Software for Consensual Cross-Platform Lipidomics

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

Exogenous Ether Lipids Predominantly Target Mitochondria

Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in ether lipid metabolism and intracellular ether lipid trafficking

Calcium electroporation and electrochemotherapy for cancer treatment:Importance of cell membrane composition investigated by lipidomics, calorimetry and in vitro efficacy

Abstract Calcium electroporation is a novel anti-cancer treatment investigated in clinical trials. We explored cell sensitivity to calcium electroporation and electroporation with bleomycin, using viability assays at different time and temperature points, as well as heat calorimetry, lipidomics, and flow cytometry. Three cell lines: HT29 (colon cancer), MDA-MB231 (breast cancer), and HDF-n (normal fibroblasts) were investigated for; (a) cell survival dependent on time of addition of drug relative to electroporation (1.2 kV/cm, 8 pulses, 99 µs, 1 Hz), at different temperatures (37 °C, 27 °C, 17 °C); (b) heat capacity profiles obtained by differential scanning calorimetry without added calcium; (c) lipid composition by mass spectrometry; (d) phosphatidylserine in the plasma membrane outer leaflet using flow cytometry. Temperature as well as time of drug administration affected treatment efficacy in HT29 and HDF-n cells, but not MDA-MB231 cells. Interestingly the HT29 cell line displayed a higher phase transition temperature (approximately 20 °C) versus 14 °C (HDF-n) and 15 °C (MDA-MB231). Furthermore the HT29 cell membranes had a higher ratio of ethers to esters, and a higher expression of phosphatidylserine in the outer leaflet. In conclusion, lipid composition and heat capacity of the membrane might influence permeabilisation of cells and thereby the effect of calcium electroporation and electrochemotherapy

Membranes by the Numbers

Many of the most important processes in cells take place on and across membranes. With the rise of an impressive array of powerful quantitative methods for characterizing these membranes, it is an opportune time to reflect on the structure and function of membranes from the point of view of biological numeracy. To that end, in this article, I review the quantitative parameters that characterize the mechanical, electrical and transport properties of membranes and carry out a number of corresponding order of magnitude estimates that help us understand the values of those parameters.Comment: 27 pages, 12 figure

Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species

Ether phospholipids are abundant membrane constituents present in electrically active tissues (e.g., heart and the brain) that play important roles in cellular function. Alterations of ether phospholipid molecular species contents are associated with a number of genetic disorders and human diseases.Herein, the power of shotgun lipidomics, in combination with high mass accuracy/high resolution mass spectrometry, was explored to identify a paired rule for the presence of isomeric ether phospholipid molecular species in cellular lipidomes. The rule predicts that if an ether phospholipid A'-B is present in a lipidome, its isomeric counterpart B'-A is also present (where the ' represents an ether linkage). The biochemical basis of this rule results from the fact that the enzymes which participate in either the sequential oxidation of aliphatic alcohols to fatty acids, or the reduction of long chain fatty acids to aliphatic alcohols (metabolic precursors of ether lipid synthesis), are not entirely selective with respect to acyl chain length or degree of unsaturation. Moreover, the enzymatic selectivity for the incorporation of different aliphatic chains into the obligatory precursor of ether lipids (i.e., 1-O-alkyl-glycero-3-phosphate) is also limited.This intrinsic amplification of the number of lipid molecular species present in biological membranes predicted by this rule and demonstrated in this study greatly expands the number of ether lipid molecular species present in cellular lipidomes. Application of this rule to mass spectrometric analyses provides predictive clues to the presence of specific molecular species and greatly expands the number of identifiable and quantifiable ether lipid species present in biological samples. Through appropriate alterations in the database, use of the paired rule increases the number of identifiable metabolites in metabolic networks, thereby facilitating identification of biomarkers presaging disease states

Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism

Comparative Lipidomics of Azole Sensitive and Resistant Clinical Isolates of Candida albicans Reveals Unexpected Diversity in Molecular Lipid Imprints

Although transcriptome and proteome approaches have been applied to determine the regulatory circuitry behind multidrug resistance (MDR) in Candida, its lipidome remains poorly characterized. Lipids do acclimatize to the development of MDR in Candida, but exactly how the acclimatization is achieved is poorly understood. In the present study, we have used a high-throughput mass spectrometry-based shotgun approach and analyzed the lipidome of genetically matched clinical azole-sensitive (AS) and -resistant (AR) isolates of C. albicans. By comparing the lipid profiling of matched isolates, we have identified major classes of lipids and determined more than 200 individual molecular lipid species among these major classes. The lipidome analysis has been statistically validated by principal component analysis. Although each AR isolate was similar with regard to displaying a high MIC to drugs, they had a distinct lipid imprint. There were some significant commonalities in the lipid profiles of these pairs, including molecular lipid species ranging from monounsaturated to polyunsaturated fatty acid-containing phosphoglycerides. Consistent fluctuation in phosphatidyl serine, mannosylinositolphosphorylceramides, and sterol esters levels indicated their compensatory role in maintaining lipid homeostasis among most AR isolates. Notably, overexpression of either CaCdr1p or CaMdr1p efflux pump proteins led to a different lipidomic response among AR isolates. This study clearly establishes the versatility of lipid metabolism in handling azole stress among various matched AR isolates. This comprehensive lipidomic approach will serve as a resource for assessing strategies aimed at disrupting the functions of Candida lipids, particularly the functional interactions between lipids and MDR determinants

Lipid metabolic perturbation is an early-onset phenotype in adult spinster mutants: a Drosophila model for lysosomal storage disorders

Intracellular accumulation of lipids and swollen dysfunctional lysosomes are linked to several neurodegenerative diseases, including lysosomal storage disorders (LSD). Detailed characterization of lipid metabolic changes in relation to the onset and progression of neurodegeneration is currently missing. We systematically analyzed lipid perturbations in spinster (spin) mutants, a Drosophila model of LSD-like neurodegeneration. Our results highlight an imbalance in brain ceramide and sphingosine in the early stages of neurodegeneration, preceding the accumulation of endomembranous structures, manifestation of altered behavior, and buildup of lipofuscin. Manipulating levels of ceramidase and altering these lipids in spin mutants allowed us to conclude that ceramide homeostasis is the driving force in disease progression and is integral to spin function in the adult nervous system. We identified 29 novel physical interaction partners of Spin and focused on the lipid carrier protein, Lipophorin (Lpp). A subset of Lpp and Spin colocalize in the brain and within organs specialized for lipid metabolism (fat bodies and oenocytes). Reduced Lpp protein was observed in spin mutant tissues. Finally, increased levels of lipid metabolites produced by oenocytes in spin mutants allude to a functional interaction between Spin and Lpp, underscoring the systemic nature of lipid perturbation in LSD

Pharmacokinetic-Pharmacodynamic Modeling of the D2 and 5-HT2A Receptor Occupancy of Risperidone and Paliperidone in Rats

A pharmacokinetic-pharmacodynamic (PK-PD) model was developed to describe the time course of brain concentration and dopamine D-2 and serotonin 5-HT2A receptor occupancy (RO) of the atypical antipsychotic drugs risperidone and paliperidone in rats. A population approach was utilized to describe the PK-PD of risperidone and paliperidone using plasma and brain concentrations and D-2 and 5-HT2A RO data. A previously published physiology- and mechanism-based (PBPKPD) model describing brain concentrations and D-2 receptor binding in the striatum was expanded to include metabolite kinetics, active efflux from brain, and binding to 5-HT2A receptors in the frontal cortex. A two-compartment model best fit to the plasma PK profile of risperidone and paliperidone. The expanded PBPKPD model described brain concentrations and D-2 and 5-HT2A RO well. Inclusion of binding to 5-HT2A receptors was necessary to describe observed brain-to-plasma ratios accurately. Simulations showed that receptor affinity strongly influences brain-to-plasma ratio pattern. Binding to both D-2 and 5-HT2A receptors influences brain distribution of risperidone and paliperidone. This may stem from their high affinity for D-2 and 5-HT2A receptors. Receptor affinities and brain-to-plasma ratios may need to be considered before choosing the best PK-PD model for centrally active drugs

A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors