{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002047","ANALYSIS_ID":"AN003334","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"Lyso-lipid induced oligodendrocytes maturation underlie restoration of optic nerve function","PROJECT_SUMMARY":"Protein hyper-deimination and deficiency of lyso-phospholipids (LPC 18:1) has been associated with the pathology of demyelinating disease in both humans and mice. We uncovered interesting biology of LPC 18:1, in which LPC 18:1 induced optic nerve function restoration through oligodendrocyte maturation and remyelination in mouse model systems. Our in vitro studies show LPC 18:1 protection against neuron-ectopic hyper-deimination and stimulation of oligodendrocyte maturation, while in vivo investigations recorded optic nerve function improvement following optic nerve injections of LPC 18:1, in contrast to LPC 18:0. Thus just a change in a single bond renders a dramatic alternation in biological function. The incorporation of isobaric C13-histidine in newly synthesized myelin proteins and quantitative proteome shifts are consistent with remyelination underlying restoration in optic nerve function. These results suggest that exogenous LPC 18:1 may provide a therapeutic avenue for stemming vision loss in demyelinating diseases.","INSTITUTE":"University of Miami","DEPARTMENT":"Ophthalmology","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy K.","ADDRESS":"1638 NW 10th Avenue, Room 706-A, Miami, FL 33136","EMAIL":"sbhattacharya@med.miami.edu","PHONE":"3054824103","DOI":"http://dx.doi.org/10.21228/M8FQ39"},

"STUDY":{"STUDY_TITLE":"Lyso-lipid induced oligodendrocytes maturation underlie restoration of optic nerve function","STUDY_SUMMARY":"Protein hyper-deimination and deficiency of lyso-phospholipids (LPC 18:1) has been associated with the pathology of demyelinating disease in both humans and mice. We uncovered interesting biology of LPC 18:1, in which LPC 18:1 induced optic nerve function restoration through oligodendrocyte maturation and remyelination in mouse model systems. Our in vitro studies show LPC 18:1 protection against neuron-ectopic hyper-deimination and stimulation of oligodendrocyte maturation, while in vivo investigations recorded optic nerve function improvement following optic nerve injections of LPC 18:1, in contrast to LPC 18:0. Thus just a change in a single bond renders a dramatic alternation in biological function. The incorporation of isobaric C13-histidine in newly synthesized myelin proteins and quantitative proteome shifts are consistent with remyelination underlying restoration in optic nerve function. These results suggest that exogenous LPC 18:1 may provide a therapeutic avenue for stemming vision loss in demyelinating diseases.","INSTITUTE":"University of Miami","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy K.","ADDRESS":"1638 NW 10th Avenue, Room 706-A, Miami, FL 33136","EMAIL":"sbhattacharya@med.miami.edu","PHONE":"3054824103","SUBMIT_DATE":"2021-12-16"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Rattus norvegicus","TAXONOMY_ID":"10116"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"OLGO_NN_20210318164109",
"Factors":{"Factor":"Olig Over"},
"Additional sample data":{"RAW_FILE_NAME":"OLGO_NN_POS_20210318164109.raw","Other":"OLGO_NN_NEG_20210326180553.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OLGO_NP",
"Factors":{"Factor":"Olig Over"},
"Additional sample data":{"RAW_FILE_NAME":"OLGO_NP_POS.raw","Other":"OLGO_NP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OLGO_NP_20210318175410",
"Factors":{"Factor":"Olig Over"},
"Additional sample data":{"RAW_FILE_NAME":"OLGO_NP_POS_20210318175410.raw","Other":"OLGO_NP_NEG_20210327020026.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OLGO_PN",
"Factors":{"Factor":"Olig Over"},
"Additional sample data":{"RAW_FILE_NAME":"OLGO_PN_POS.raw","Other":"OLGO_PN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OLGO_PN_20210318202010",
"Factors":{"Factor":"Olig Over"},
"Additional sample data":{"RAW_FILE_NAME":"OLGO_PN_POS_20210318202010.raw","Other":"OLGO_PN_NEG_20210326191854.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OLGO_PP",
"Factors":{"Factor":"Olig Over"},
"Additional sample data":{"RAW_FILE_NAME":"OLGO_PP_POS.raw","Other":"OLGO_PP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OLGO_PP_20210319014845",
"Factors":{"Factor":"Olig Over"},
"Additional sample data":{"RAW_FILE_NAME":"OLGO_PP_POS_20210319014845.raw","Other":"OLGO_PP_NEG_20210326233426.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NNN_2",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NNN_2_POS.raw","Other":"Olig_NNN_2_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NNN_2_20210317100746",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NNN_2_POS_20210317100746.raw","Other":"Olig_NNN_2_NEG_20210318023305.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NNP_2",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NNP_2_POS.raw","Other":"Olig_NNP_2_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NNP_2_20210317074144",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NNP_2_POS_20210317074144.raw","Other":"Olig_NNP_2_NEG_20210317161247.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NNP_3",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NNP_3_POS.raw","Other":"Olig_NNP_3_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NNP_3_20210317032612",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NNP_3_POS_20210317032612.raw","Other":"Olig_NNP_3_NEG_20210318045901.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NPN_2",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NPN_2_POS.raw","Other":"Olig_NPN_2_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_NPN_2_20210317010010",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_NPN_2_POS_20210317010010.raw","Other":"Olig_NPN_2_NEG_20210318000706.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_PNN_2",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_PNN_2_POS.raw","Other":"Olig_PNN_2_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_PNN_2_20210317112047",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_PNN_2_POS_20210317112047.raw","Other":"Olig_PNN_2_NEG_20210318012005.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_PPP_2",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_PPP_2_POS.raw","Other":"Olig_PPP_2_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Olig_PPP_2_20210317043913",
"Factors":{"Factor":"Olig siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"Olig_PPP_2_POS_20210317043913.raw","Other":"Olig_PPP_2_NEG_20210317195142.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_NN",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_NN_POS.raw","Other":"OPCO_NN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_NN_20210318220941",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_NN_POS_20210318220941.raw","Other":"OPCO_NN_NEG_20210327031329.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_NP",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_NP_POS.raw","Other":"OPCO_NP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_NP_20210319003544",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_NP_POS_20210319003544.raw","Other":"OPCO_NP_NEG_20210327004725.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_PN",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_PN_POS.raw","Other":"OPCO_PN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_PN_20210318232241",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_PN_POS_20210318232241.raw","Other":"OPCO_PN_NEG_20210326214456.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_PP",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_PP_POS.raw","Other":"OPCO_PP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPCO_PP_20210318190709",
"Factors":{"Factor":"OPC Over"},
"Additional sample data":{"RAW_FILE_NAME":"OPCO_PP_POS_20210318190709.raw","Other":"OPCO_PP_NEG_20210326203155.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_NNN_1",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_NNN_1_POS.raw","Other":"OPC_NNN_1_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_NNN_1_20210317085445",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_NNN_1_POS_20210317085445.raw","Other":"OPC_NNN_1_NEG_20210317210441.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_NNP_1",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_NNP_1_POS.raw","Other":"OPC_NNP_1_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_NNP_1_20210317123348",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_NNP_1_POS_20210317123348.raw","Other":"OPC_NNP_1_NEG_20210317183845.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_NPN_1",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_NPN_1_POS.raw","Other":"OPC_NPN_1_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_NPN_1_20210317021311",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_NPN_1_POS_20210317021311.raw","Other":"OPC_NPN_1_NEG_20210317172546.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_PNN_1",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_PNN_1_POS.raw","Other":"OPC_PNN_1_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_PNN_1_20210317134649",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_PNN_1_POS_20210317134649.raw","Other":"OPC_PNN_1_NEG_20210317225408.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_PPP_1",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_PPP_1_POS.raw","Other":"OPC_PPP_1_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"OPC_PPP_1_20210317055214",
"Factors":{"Factor":"OPC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"OPC_PPP_1_POS_20210317055214.raw","Other":"OPC_PPP_1_NEG_20210318034601.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_over_NN",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_over_NN_POS.raw","Other":"repRGC_over_NN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_over_NP",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_over_NP_POS.raw","Other":"repRGC_over_NP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_over_PN",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_over_PN_POS.raw","Other":"repRGC_over_PN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_over_PP",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_over_PP_POS.raw","Other":"repRGC_over_PP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_over_NN",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_over_NN_POS.raw","Other":"RGC_over_NN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_over_NP",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_over_NP_POS.raw","Other":"RGC_over_NP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_over_PN",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_over_PN_POS.raw","Other":"RGC_over_PN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_over_PP",
"Factors":{"Factor":"RGC Over"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_over_PP_POS.raw","Other":"RGC_over_PP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_si_NNN",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_si_NNN_POS.raw","Other":"repRGC_si_NNN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_si_NNP",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_si_NNP_POS.raw","Other":"repRGC_si_NNP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_si_NPN",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_si_NPN_POS.raw","Other":"repRGC_si_NPN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_si_PNN",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_si_PNN_POS.raw","Other":"repRGC_si_PNN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"repRGC_si_PPP",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"repRGC_si_PPP_POS.raw","Other":"repRGC_si_PPP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_si_NNN",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_si_NNN_POS.raw","Other":"RGC_si_NNN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_si_NNP",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_si_NNP_POS.raw","Other":"RGC_si_NNP_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_si_NPN",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_si_NPN_POS.raw","Other":"RGC_si_NPN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_si_PNN",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_si_PNN_POS.raw","Other":"RGC_si_PNN_NEG.raw"}
},
{
"Subject ID":"-",
"Sample ID":"RGC_si_PPP",
"Factors":{"Factor":"RGC siRNA"},
"Additional sample data":{"RAW_FILE_NAME":"RGC_si_PPP_POS.raw","Other":"RGC_si_PPP_NEG.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"RGC cell isolation. Primary retinal ganglion cells (RGCs) were isolated according to the two-step immunopanning protocol described in a previous study (Dvoriantchikova, Degterev et al., 2014). Briefly, whole retinas of 10 postnatal day 11-12 pups were incubated in papain solution (16.5 U/mL; Worthington Biochemical, LS003127) for 30 minutes. Macrophaged and endothelial cells were removed from the cell suspension by panning with anti-macrophage antiserum (Accurate Chemical, AIA31240). RGCs were bound to the panning plates containing the antibody against Thy1.2 (VWR, 102646-060) and were then released by incubation with trypsin solution (Sigma, T9935). RGCs were plated in a plate coated with poly-d-lysine (Sigma, P6407) and Laminin (Sigma, L-6274). RGCs were either not treated (addition of RGC growth media), treated with LPC 18:0 at 10 µM in RGC growth media, or treated with LPC 18:1 at 10 µM in RGC growth media. RGCs were incubated for 24 hours and harvested the next day. OPC cell culture. Oligodendrocyte Precursor Cells (OPC) were purchased from ScienceCell Research Laboratories (R1600, Carlsbad, CA 92008) and cultured according to manufacturer’s recommendations. In brief, plates were coated with poly-d-lysine (Sigma, P6407; 2 µg/cm2) and incubated overnight at 37ºC. OPC growth media was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC growth supplement (ScienceCell Research Laboratories, 1652) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). OPC differentiation medium was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC differentiation supplement (ScienceCell Research Laboratories, 1672), fetal bovine serum (ScienceCell Research Laboratories, 0005) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). Poly-d-lysine coated plate was washed twice with sterile water, OPC growth media was added to each well and plate was placed in incubator to equilibrate for 15 minutes (37ºC and 5% CO2). Frozen vial containing OPCs was warmed in 37ºC water bath and gently rotated to equally suspend cells. 15,000 cells/cm2 were seeded and left overnight (~16 to ~18 hours) in incubator. Media was changed daily for 2 days. OPCs were either not treated (addition of OPC growth media), differentiated (addition of OPC differentiation media), treated with LPC 18:0 at 10 µM in OPC growth media, or treated with LPC 18:1 at 10 µM in OPC growth media. OPCs were incubated for 24 hours and harvested the next day. OPCs and oligodendrocytes were further validated using mRNA analysis following published methods (Jäkel, Agirre et al., 2019).","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"siRNA and overexpression. Cell transfection was carried using the INTERFERin transfection kit following manufacture’s recommendations (Polyplus Transfection, 89129-930). siRNA for LPCAT1, PLA2G4C, and LIPC were purchased from Ambion (Ambion, s102346, s107315, s67780 respectively), LPGAT1 and LIPC overexpression constructs were purchased from Genecopoeia (Genecopoeia, EX-mm15104-M61, EX-mm03119-M61). In brief, cells were transfected with 2 nM of siRNA or DNA in serum free cell culture media. INTERFERin reagent was added to the mix incubated at room temperature for 10 minutes and added to newly changed cell culture media for each well. Cells were incubated for 48 to 96 hours before harvesting."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"15 µg of total protein from homogenate sample were added to 4 times the volume of acetone at 20°C and incubated at 20°C overnight. Samples were centrifuged at 21,000 g at 4°C for 30 minutes (Thermo Fisher Megafuge 8R). Supernatant was discarded and pellet (very small) was air dried for 10 minutes. The pellet was re-suspended, denatured and reduced with 6 M urea, 10 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature. Following denaturing and reduction, the re-suspended pellet was alkylated with 15 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 minutes while maintained in darkness. Alkylation reaction was quench using 20 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature while kept in darkness. Sample was diluted using 50 mM ammonium bicarbonate to contain 1 M urea. Sample was digested using either trypsin or chymotrypsin (Promega, V5111, V106A) at a 1:30 (w/w) ratio of enzyme to protein. Digestion was incubated overnight at 37°C. Reaction was terminated using 50% formic acid at a 5:100 (v/v) ratio of formic acid to sample volume. Samples were stored at -20°C or immediately desalted. Samples were desalted using the Pierce Graphite Spin Columns (Thermo, 88302) following manufacturer’s recommendations. Samples were then evaporated using the CentriVap Concentrator system (Labconco, Kansas City, MO 64132-2696) and resuspended in 30 µl of protein resuspension solution [2% (v/v) acetonitrile, 0.1% (v/v) formic acid in mass spectrometry grade water]. Lipids were harvested by adding 400 µl of 1:1 v/v methanol/chloroform mix to 200 µl of cell culture lysate. Lysate was vortexed and incubated in ice for 5 minutes, followed by the addition of 350 µl of chloroform. Lysate was centrifuged at 18,000g for 15 minutes at 4ºC. The organic layer containing lipids (bottom layer) was transferred to a new tube and desiccated using the CentriVap Concentrator system. Lipids were resuspended in 50 µl of lipid resuspension solution [50% v/v isopropyl alcohol and 50% v/v acetonitrile]."},

"CHROMATOGRAPHY":{"INSTRUMENT_NAME":"Thermo Accela 600","COLUMN_NAME":"Thermo Acclaim C30 (150 x 2.1mm,3um)","CHROMATOGRAPHY_TYPE":"Reversed phase"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","MS_COMMENTS":"Lipids were identified using LipidSearch software v. 4.1.","ION_MODE":"UNSPECIFIED"},

"MS_METABOLITE_DATA":{
"Units":"μg/ml",

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