{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001288","ANALYSIS_ID":"AN002141","VERSION":"1","CREATED_ON":"December 17, 2019, 7:20 am"},

"PROJECT":{"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"},

"STUDY":{"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","EMAIL":"efahy@ucsd.edu","PHONE":"858-534-4076","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."},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"S1",
"Factors":{"Fraction":"Nucleus","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S2",
"Factors":{"Fraction":"Nucleus","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S3",
"Factors":{"Fraction":"Nucleus","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S4",
"Factors":{"Fraction":"Nucleus","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S5",
"Factors":{"Fraction":"Nucleus","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S6",
"Factors":{"Fraction":"Nucleus","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S7",
"Factors":{"Fraction":"Mitochondria","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S8",
"Factors":{"Fraction":"Mitochondria","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S9",
"Factors":{"Fraction":"Mitochondria","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S10",
"Factors":{"Fraction":"Mitochondria","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S11",
"Factors":{"Fraction":"Mitochondria","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S12",
"Factors":{"Fraction":"Mitochondria","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S13",
"Factors":{"Fraction":"ER","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S14",
"Factors":{"Fraction":"ER","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S15",
"Factors":{"Fraction":"ER","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S16",
"Factors":{"Fraction":"ER","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S17",
"Factors":{"Fraction":"ER","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S18",
"Factors":{"Fraction":"ER","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S19",
"Factors":{"Fraction":"Plasma membrane","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S20",
"Factors":{"Fraction":"Plasma membrane","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S21",
"Factors":{"Fraction":"Plasma membrane","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S22",
"Factors":{"Fraction":"Plasma membrane","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S23",
"Factors":{"Fraction":"Plasma membrane","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S24",
"Factors":{"Fraction":"Plasma membrane","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S25",
"Factors":{"Fraction":"Dense microsomes","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S26",
"Factors":{"Fraction":"Dense microsomes","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S27",
"Factors":{"Fraction":"Dense microsomes","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S28",
"Factors":{"Fraction":"Dense microsomes","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S29",
"Factors":{"Fraction":"Dense microsomes","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S30",
"Factors":{"Fraction":"Dense microsomes","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S31",
"Factors":{"Fraction":"Cytosol","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S32",
"Factors":{"Fraction":"Cytosol","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S33",
"Factors":{"Fraction":"Cytosol","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S34",
"Factors":{"Fraction":"Cytosol","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S35",
"Factors":{"Fraction":"Cytosol","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S36",
"Factors":{"Fraction":"Cytosol","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S37",
"Factors":{"Fraction":"Whole cells","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S38",
"Factors":{"Fraction":"Whole cells","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S39",
"Factors":{"Fraction":"Whole cells","Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"S40",
"Factors":{"Fraction":"Whole cells","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S41",
"Factors":{"Fraction":"Whole cells","Treatment":"KLA"}
},
{
"Subject ID":"-",
"Sample ID":"S42",
"Factors":{"Fraction":"Whole cells","Treatment":"KLA"}
}
],
"COLLECTION":{"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_SUMMARY":"Subcellular fractionation procedures are described in detail in the publication doi: 10.1194/jlr.M008748"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The various extraction procedures and lipidomic analyses for each lipid class are described in the publication doi: 10.1194/jlr.M008748"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"See publication for details doi: 10.1194/jlr.M008748","CHROMATOGRAPHY_TYPE":"Reversed phase/Normal phase","INSTRUMENT_NAME":"Several","COLUMN_NAME":"Several"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Several","INSTRUMENT_TYPE":"Several","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"See publication for details. doi: 10.1194/jlr.M008748"},

"MS_METABOLITE_DATA":{
"Units":"pmoles per mg protein",

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}

}