scholarly journals Quantitative evaluation of a high resolution lipidomics platform

2019 ◽  
Author(s):  
Juan Liu ◽  
Xiaojing Liu ◽  
Zhengtao Xiao ◽  
Jason W. Locasale
Keyword(s):  
Fatty Acid ◽  
High Resolution ◽  
Mammalian Cells ◽  
De Novo ◽  
High Resolution Mass Spectrometry ◽  
Methyl Tert Butyl Ether ◽  
Ovarian Cancer Cells ◽  
General Importance ◽  
Lipid Species ◽  
Polar Metabolites

AbstractGiven the general importance of lipids in health and disease, there is a need for efficient technology that broadly profiles and quantitates the lipid composition of complex mixtures. In this study, we developed and quantitatively evaluated a platform that simultaneously profiles both lipids and polar metabolites from the same sample. This method was achieved by using a methyl tert-butyl ether (MTBE) extraction and employing two liquid chromatography methods coupled with high resolution mass spectrometry (LC-HRMS). This workflow enabled detection and semi-quantitation of over 300 polar metabolites as well as over 300 lipids with comprehensive coverage of diverse chemical classes. Using cultured mammalian cells as an example, we report the quantitative properties of the platform including the sensitivity and linear range. The lipidomics strategy was further applied to characterize changes to lipid metabolism upon treatment with metformin to human ovarian cancer cells. Of the 256 detected lipids, 99 lipids (39%) significantly increased, 11 lipids (4%) were significantly reduced and 146 lipids (57%) remain unchanged in metformin-treated cells. Stable isotope tracing of carbon into lipids using [13C6]-glucose further measured the contribution of de novo fatty acid synthesis to the total fatty acid pool. In summary, the platform enabled the semi-quantitative assessment of hundreds of lipid species and associated carbon incorporation from glucose in a high throughput manner.

scholarly journals A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 118 ◽  
Author(s):  
Magdalena Topolska ◽  
Fernando Martínez-Montañés ◽  
Christer S. Ejsing
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acid ◽  
High Resolution ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
High Resolution Mass Spectrometry ◽  
Enzymatic Process ◽  
Product Specificity ◽  
High Resolution Mass ◽  
Resolution Mass

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of de novo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.

scholarly journals The Pharmacological or Genetic Blockade of Endogenous De Novo Fatty Acid Synthesis Does Not Increase the Uptake of Exogenous Lipids in Ovarian Cancer Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Thomas W. Grunt ◽  
Lisa Lemberger ◽  
Ramón Colomer ◽  
María Luz López−Rodríguez ◽  
Renate Wagner
Keyword(s):  
Ovarian Cancer ◽  
Fatty Acid ◽  
De Novo ◽  
Lipid Production ◽  
Growth Arrest ◽  
High Energy ◽  
Cancer Drug ◽  
Fatty Acid Binding Proteins ◽  
Ovarian Cancer Cells ◽  
Fatty Acid Binding

Ovarian cancer(OC) is a serious threat to women worldwide. Peritoneal dissemination, ascites and omental metastasis are typical features for disease progression, which occurs in a micro-environment that is rich in high-energy lipids. OC cells require high amounts of lipids for survival and growth. Not only do they import lipids from the host, they also produce lipids de novo. Inhibitors of fatty acid(FA) synthase(FASN) – the rate-limiting enzyme of endogenous FA synthesis that is overexpressed in OC – induce growth-arrest and apoptosis, rendering them promising candidates for cancer drug development. However, cancer researchers have long hypothesized that the lipid deficiency caused by FASN inhibition can be circumvented by increasing the uptake of exogenous lipids from the host, which would confer resistance to FASN inhibitors. In contrast to a very recent report in colorectal cancer, we demonstrate in OC cells (A2780, OVCAR3, SKOV3) that neither FASN inhibitors (G28UCM, Fasnall) nor FASN-specific siRNAs can stimulate a relief pathway leading to enhanced uptake of extrinsic FAs or low density lipoproteins (LDLs). Instead, we observed that the growth-arrest due to FASN inhibition or FASN knock-down was associated with significant dose- and time-dependent reduction in the uptake of fluorescently labeled FAs and LDLs. Western blotting showed that the expression of the FA receptor CD36, the LDL receptor(LDLR) and the lipid transport proteins fatty acid binding proteins 1–9 (FABP1–9) was not affected by the treatment. Next, we compared experimental blockade of endogenous lipid production with physiologic depletion of exogenous lipids. Lipid-free media, similar to FASN inhibitors, caused growth-arrest. Although lipid-depleted cells have diminished amounts of CD36, LDLR and FABPs, they can still activate a restorative pathway that causes enhanced import of fluorophore-labeled FAs and LDLs. Overall, our data show that OC cells are strictly lipid-depend and exquisitely sensitive to FASN inhibitors, providing a strong rationale for developing anti-FASN strategies for clinical use against OC.

scholarly journals A combined flow injection/reversed phase chromatography – high resolution mass spectrometry workflow for accurate absolute lipid quantification with 13C- internal standards

2020 ◽  
Author(s):  
Harald Schoeny ◽  
Evelyn Rampler ◽  
Yasin El Abiead ◽  
Felina Hildebrand ◽  
Olivia Zach ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Flow Injection ◽  
High Resolution Mass Spectrometry ◽  
Reversed Phase ◽  
Internal Standards ◽  
High Resolution Mass ◽  
Lipid Species ◽  
Species Specific ◽  
Resolution Mass

ABSTRACTWe propose a fully automated novel workflow for lipidomics based on flow injection-followed by liquid chromatography high resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed phase LC-HRMS with absolute quantification as obtained by a high number of lipid species-specific- and/or retention time (RT) matched/class-specific calibrants. The lipidome of 13C labelled yeast (LILY) provided a cost efficient, large panel of internal standards covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). In order to exploit the full potential of isotopically enriched biomass, LILY was absolutely quantified on demand via reversed isotope dilution analysis using FI-HRMS. Subsequent LC-HRMS analysis integrated different calibration strategies including lipid species-specific standards for >90 lipids. Extensive measures on quality control allowed to rank the calibration strategies and to automatically selected the calibration strategy of highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis pipeline (identification and quantification in separate analytical runs) and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level with an analytical run-time of less than 25 min per sample.TOC

Determination of fatty acid methyl esters in cosmetic castor oils by flow injection-electrospray ionization-high-resolution mass spectrometry

2018 ◽  
Vol 40 (3) ◽  
pp. 295-302 ◽  
Author(s):  
M. A. Arroyo Negrete ◽  
K. Wrobel ◽  
F. J. Acevedo Aguilar ◽  
E. Yanez Barrientos ◽  
A. R. Corrales Escobosa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acid ◽  
High Resolution ◽  
Electrospray Ionization ◽  
Fatty Acid Methyl Esters ◽  
High Resolution Mass Spectrometry ◽  
Methyl Esters ◽  
Acid Methyl ◽  
Resolution Mass

scholarly journals Single-Step Extraction Coupled with Targeted HILIC-MS/MS Approach for Comprehensive Analysis of Human Plasma Lipidome and Polar Metabolome

Metabolites ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 495
Author(s):  
Jessica Medina ◽  
Vera van der Velpen ◽  
Tony Teav ◽  
Yann Guitton ◽  
Hector Gallart-Ayala ◽  
...  
Keyword(s):  
Human Plasma ◽  
Large Scale ◽  
Lipid Extraction ◽  
Single Step ◽  
Methyl Tert Butyl Ether ◽  
Method Performance ◽  
Simultaneous Extraction ◽  
Lipid Species ◽  
Polar Metabolites ◽  
Complex Lipids

Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, we evaluated the performance of single-step sample preparation methods for simultaneous extraction of the complex lipidome and polar metabolome from human plasma. The method performance was evaluated using high-coverage Hydrophilic Interaction Liquid Chromatography-ESI coupled to tandem mass spectrometry (HILIC-ESI-MS/MS) methodology targeting a panel of 1159 lipids and 374 polar metabolites. The criteria used for method evaluation comprised protein precipitation efficiency, and relative MS signal abundance and repeatability of detectable lipid and polar metabolites in human plasma. Among the tested methods, the isopropanol (IPA) and 1-butanol:methanol (BUME) mixtures were selected as the best compromises for the simultaneous extraction of complex lipids and polar metabolites, allowing for the detection of 584 lipid species and 116 polar metabolites. The extraction with IPA showed the greatest reproducibility with the highest number of lipid species detected with the coefficient of variation (CV) < 30%. Besides this difference, both IPA and BUME allowed for the high-throughput extraction and reproducible measurement of a large panel of complex lipids and polar metabolites, thus warranting their application in large-scale human population studies.

Following De Novo Triglyceride Dynamics in Ovaries of Aedes Aegypti Mosquitoes

2021 ◽  
Author(s):  
Lilian Valadares Tose ◽  
Chad R. Weisbrod ◽  
Veronika Michalkova ◽  
Marcela Nouzova ◽  
Fernando G. Noriega ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
De Novo ◽  
High Resolution Mass Spectrometry ◽  
Building Blocks ◽  
High Mass ◽  
Metabolic Labeling ◽  
Deuterated Water ◽  
Biochemical Basis ◽  
Time Dynamics ◽  
Lipid Species

Abstract Understanding the molecular and biochemical basis of egg development is a central topic in mosquito reproductive biology. Lipids are a major source of energy and building blocks for the developing ovarian follicles. Ultra-High Resolution Mass Spectrometry (UHRMS) combined with in vivo metabolic labeling of follicle lipids with deuterated water (2H2O) can provide unequivocal identification of de novo lipid species during ovarian development. In the present study, we followed de novo triglyceride (TG) dynamics during the ovarian previtellogenic (PVG) stage (2-7 days post-eclosion) of female adult Aedes aegypti. The incorporation of stable isotopes from the diet was evaluated using liquid chromatography (LC) in tandem with the high accuracy (<0.3 ppm) and high mass resolution (over 1M) of a 14.5 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (14.5 T FT-ICR MS) equipped with hexapolar detection. LC-UHRMS provides effective lipid class separation and chemical formula identification based on the isotopic fine structure. The monitoring of stable isotope incorporation into de novo incorporated TGs suggests that ovarian lipids are consumed or recycled during the PVG stage, with variable time dynamics. These results provide further evidence of the complexity of the molecular mechanism of follicular lipid dynamics during oogenesis in mosquitoes.

scholarly journals Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors

2015 ◽  
Vol 21 (2) ◽  
pp. 111-116 ◽  
Author(s):  
Philip J. Hopcroft ◽  
David I. Fisher
Keyword(s):  
Fatty Acid ◽  
Mammalian Cells ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Structure Activity ◽  
Malonyl Coa ◽  
Scale Normalization ◽  
Development Structure ◽  
Screening Cascade

The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography–mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure–activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z′ of >0.6, and has high reproducibility of EC50 values.

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