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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001288 |
| Study Title | Subcellular organelle lipidomics in TLR-4-activated macrophages |
| Study 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 | LIPID MAPS |
| Department | Multiple |
| Laboratory | Multiple |
| Last Name | Fahy |
| First Name | Eoin |
| Address | 9500 Gilman, La Jolla, CA, 92093, USA |
| efahy@ucsd.edu | |
| Phone | 858-534-4076 |
| Submit Date | 2019-12-17 |
| Publications | Andreyev 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 Detail | LC-MS |
| Release Date | 2020-01-22 |
| Release Version | 1 |
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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 |
