Summary of study ST001288

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000870. The data can be accessed directly via it's Project DOI: 10.21228/M8796F This work is supported by NIH grant, U2C- DK119886.

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Study IDST001288
Study TitleSubcellular organelle lipidomics in TLR-4-activated macrophages
Study SummaryLipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo2-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g. increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation.
Institute
LIPID MAPS
DepartmentMultiple
LaboratoryMultiple
Last NameFahy
First NameEoin
Address9500 Gilman, La Jolla, CA, 92093, USA
Emailefahy@ucsd.edu
Phone858-534-4076
Submit Date2019-12-17
PublicationsAndreyev AY, Fahy E, Guan Z, Kelly S, Li X, McDonald JG, Milne S, Myers D, Park H, Ryan A, Thompson BM, Wang E, Zhao Y, Brown HA, Merrill AH, Raetz CR, Russell DW, Subramaniam S, Dennis EA. Subcellular organelle lipidomics in TLR-4-activated macrophages. J Lipid Res. 2010 Sep;51(9):2785-97. doi: 10.1194/jlr.M008748. Epub 2010 Jun 23. PMID: 20574076; PMCID: PMC2918461.
Analysis Type DetailLC-MS
Release Date2020-01-22
Release Version1
Eoin Fahy Eoin Fahy
https://dx.doi.org/10.21228/M8796F
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000870
Project DOI:doi: 10.21228/M8796F
Project Title:Subcellular organelle lipidomics in TLR-4-activated macrophages
Project Summary:Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo2-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g. increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation.
Institute:University of California, San Diego
Department:Bioengineering
Last Name:Fahy
First Name:Eoin
Address:9500 Gilman, La Jolla, CA, 92093, USA
Email:efahy@ucsd.edu
Phone:858-534-4076

Subject:

Subject ID:SU001360
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Fraction Treatment
SA093638S32Cytosol Control
SA093639S31Cytosol Control
SA093640S33Cytosol Control
SA093641S36Cytosol KLA
SA093642S34Cytosol KLA
SA093643S35Cytosol KLA
SA093644S25Dense microsomes Control
SA093645S26Dense microsomes Control
SA093646S27Dense microsomes Control
SA093647S29Dense microsomes KLA
SA093648S30Dense microsomes KLA
SA093649S28Dense microsomes KLA
SA093650S15ER Control
SA093651S14ER Control
SA093652S13ER Control
SA093653S18ER KLA
SA093654S17ER KLA
SA093655S16ER KLA
SA093656S9Mitochondria Control
SA093657S8Mitochondria Control
SA093658S7Mitochondria Control
SA093659S11Mitochondria KLA
SA093660S10Mitochondria KLA
SA093661S12Mitochondria KLA
SA093662S1Nucleus Control
SA093663S3Nucleus Control
SA093664S2Nucleus Control
SA093665S5Nucleus KLA
SA093666S6Nucleus KLA
SA093667S4Nucleus KLA
SA093668S20Plasma membrane Control
SA093669S19Plasma membrane Control
SA093670S21Plasma membrane Control
SA093671S22Plasma membrane KLA
SA093672S23Plasma membrane KLA
SA093673S24Plasma membrane KLA
SA093674S39Whole cells Control
SA093675S38Whole cells Control
SA093676S37Whole cells Control
SA093677S42Whole cells KLA
SA093678S41Whole cells KLA
SA093679S40Whole cells KLA
Showing results 1 to 42 of 42

Collection:

Collection ID:CO001354
Collection Summary:Immortalized mouse macrophage-like RAW264.7 cells were obtained from the ATCC (catalog no. TIB-71). DMEM (catalog no. 10-013) and PBS (catalog no. 21-031-CV) were from Mediatech. Fetal calf serum with low endotoxin content was from Hyclone (SH30071.03 ANG19242). KLA and lipid standards were from Avanti Polar Lipids. Iodixanol (OptiPrep from Axis-Shield) was obtained from Sigma-Aldrich. Solvents were chromatography-grade and purchased from OmniSolv. All other reagents/kits were from Sigma-Aldrich. Aqueous solutions were prepared using distilled-deionized water (catalog no. 25-055-CV) from Mediatech. Isolation media were prepared K+- and Na+-free; pH was adjusted by addition of Tris base (TRIZMA). Cells were maintained, treated, and fractionated as previously described (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2830848/). Briefly, RAW264.7 cells were maintained between passages 4 and 24 at 37°C and 10% CO2. The medium was composed of high glucose- and l-glutamine-containing DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. For an experiment, five T-150 flasks of cells were plated at a density of 3.6 × 107 cells/flask in 24 ml of medium. At 24 h after plating, cells were treated with vehicle or 100 ng/ml KLA for another 24 h followed by subcellular fractionation.
Sample Type:Macrophages

Treatment:

Treatment ID:TR001375
Treatment Summary:Subcellular fractionation procedures are described in detail in the publication doi: 10.1194/jlr.M008748

Sample Preparation:

Sampleprep ID:SP001368
Sampleprep Summary:The various extraction procedures and lipidomic analyses for each lipid class are described in the publication doi: 10.1194/jlr.M008748

Combined analysis:

Analysis ID AN002141
Analysis type MS
Chromatography type Reversed phase/Normal phase
Chromatography system Several
Column Several
MS Type ESI
MS instrument type Several
MS instrument name Several
Ion Mode UNSPECIFIED
Units pmoles per mg protein

Chromatography:

Chromatography ID:CH001567
Chromatography Summary:See publication for details doi: 10.1194/jlr.M008748
Instrument Name:Several
Column Name:Several
Chromatography Type:Reversed phase/Normal phase

MS:

MS ID:MS001993
Analysis ID:AN002141
Instrument Name:Several
Instrument Type:Several
MS Type:ESI
MS Comments:See publication for details. doi: 10.1194/jlr.M008748
Ion Mode:UNSPECIFIED
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