#METABOLOMICS WORKBENCH kcontrep_20220812_161222 DATATRACK_ID:3404 STUDY_ID:ST002257 ANALYSIS_ID:AN003687 PROJECT_ID:PR001444 VERSION 1 CREATED_ON August 15, 2022, 10:35 am #PROJECT PR:PROJECT_TITLE Lipidomic profiling reveals age-dependent changes in complex plasma membrane PR:PROJECT_TITLE lipids that regulate neural stem cell aging PR:PROJECT_SUMMARY Study of lipid changes with age in NSC PR:INSTITUTE Stanford University PR:LAST_NAME Contrepois PR:FIRST_NAME Kevin PR:ADDRESS 300 Pasteur Dr 94305 Stanford PR:EMAIL kcontrep@stanford.edu PR:PHONE 650-664-7325 #STUDY ST:STUDY_TITLE Lipidomic profiling reveals age-dependent changes in complex plasma membrane ST:STUDY_TITLE lipids that regulate neural stem cell aging ST:STUDY_SUMMARY The aging brain exhibits a decline in the regenerative populations of neural ST:STUDY_SUMMARY stem cells (NSCs), which may underlie age-associated defects in sensory and ST:STUDY_SUMMARY cognitive functions1-4 . While mechanisms that restore old NSC function have ST:STUDY_SUMMARY started to be identified5-9 , the role of lipids – especially complex lipids ST:STUDY_SUMMARY – in NSC aging remains largely unclear. Using lipidomic profiling by mass ST:STUDY_SUMMARY spectrometry, we identify age-related lipidomic signatures in young and old ST:STUDY_SUMMARY quiescent NSCs in vitro and in vivo. These analyses reveal drastic changes in ST:STUDY_SUMMARY several complex membrane lipid classes, including phospholipids and ST:STUDY_SUMMARY sphingolipids in old NSCs. Moreover, polyunsaturated fatty acids (PUFAs) ST:STUDY_SUMMARY strikingly increase across complex lipid classes in quiescent NSCs during aging. ST:STUDY_SUMMARY Lipidomic profiling of isolated plasma membrane vesicles shows that agerelated ST:STUDY_SUMMARY differences in complex lipid levels and side chain composition are largely ST:STUDY_SUMMARY occurring in plasma membrane lipids. Experimentally, we find that aging is ST:STUDY_SUMMARY accompanied by modifications in membrane biophysical properties, with a decrease ST:STUDY_SUMMARY in plasma membrane order in old quiescent NSCs in vitro and in vivo. To ST:STUDY_SUMMARY determine the functional role of plasma membrane lipids in aging NSCs, we ST:STUDY_SUMMARY perform genetic and supplementations studies. Knockout of Mboat2, which encodes ST:STUDY_SUMMARY a phospholipid acyltransferase, exacerbates age-related lipidomic changes in old ST:STUDY_SUMMARY quiescent NSCs and impedes their ability to activate. As Mboat2 expression ST:STUDY_SUMMARY declines with age, Mboat2 deficiency may drive NSC decline during aging. ST:STUDY_SUMMARY Interestingly, supplementation of plasma membrane lipids derived from young NSCs ST:STUDY_SUMMARY boosts the ability of old quiescent NSCs to activate. Our work could lead to ST:STUDY_SUMMARY lipid-based strategies for restoring the regenerative potential of NSCs in old ST:STUDY_SUMMARY individuals, which has important implications to counter brain decline during ST:STUDY_SUMMARY aging. ST:INSTITUTE Stanford University ST:LAST_NAME Contrepois ST:FIRST_NAME Kevin ST:ADDRESS 300 Pasteur Dr 94305 Stanford ST:EMAIL kcontrep@stanford.edu ST:PHONE 650-664-7325 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - XZ1 Cell type:Activated NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ1; RAW_FILE_NAME=nRPLC_XZ1 SUBJECT_SAMPLE_FACTORS - XZ10 Cell type:Activated NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ10; RAW_FILE_NAME=nRPLC_XZ10 SUBJECT_SAMPLE_FACTORS - XZ11 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ11; RAW_FILE_NAME=nRPLC_XZ11 SUBJECT_SAMPLE_FACTORS - XZ12 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ12; RAW_FILE_NAME=nRPLC_XZ12 SUBJECT_SAMPLE_FACTORS - XZ13 Cell type:Activated NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ13; RAW_FILE_NAME=nRPLC_XZ13 SUBJECT_SAMPLE_FACTORS - XZ14 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ14; RAW_FILE_NAME=nRPLC_XZ14 SUBJECT_SAMPLE_FACTORS - XZ15 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ15; RAW_FILE_NAME=nRPLC_XZ15 SUBJECT_SAMPLE_FACTORS - XZ16 Cell type:Activated NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ16; RAW_FILE_NAME=nRPLC_XZ16 SUBJECT_SAMPLE_FACTORS - XZ17 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ17; RAW_FILE_NAME=nRPLC_XZ17 SUBJECT_SAMPLE_FACTORS - XZ18 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ18; RAW_FILE_NAME=nRPLC_XZ18 SUBJECT_SAMPLE_FACTORS - XZ19 Cell type:Activated NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ19; RAW_FILE_NAME=nRPLC_XZ19 SUBJECT_SAMPLE_FACTORS - XZ2 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ2; RAW_FILE_NAME=nRPLC_XZ2 SUBJECT_SAMPLE_FACTORS - XZ20 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ20; RAW_FILE_NAME=nRPLC_XZ20 SUBJECT_SAMPLE_FACTORS - XZ21 Cell type:Activated NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ21; RAW_FILE_NAME=nRPLC_XZ21 SUBJECT_SAMPLE_FACTORS - XZ22 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ22; RAW_FILE_NAME=nRPLC_XZ22 SUBJECT_SAMPLE_FACTORS - XZ23 Cell type:Activated NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ23; RAW_FILE_NAME=nRPLC_XZ23 SUBJECT_SAMPLE_FACTORS - XZ24 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ24; RAW_FILE_NAME=nRPLC_XZ24 SUBJECT_SAMPLE_FACTORS - XZ25 Cell type:Activated NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ25; RAW_FILE_NAME=nRPLC_XZ25 SUBJECT_SAMPLE_FACTORS - XZ26 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ26; RAW_FILE_NAME=nRPLC_XZ26 SUBJECT_SAMPLE_FACTORS - XZ27 Cell type:Activated NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ27; RAW_FILE_NAME=nRPLC_XZ27 SUBJECT_SAMPLE_FACTORS - XZ28 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ28; RAW_FILE_NAME=nRPLC_XZ28 SUBJECT_SAMPLE_FACTORS - XZ29 Cell type:Activated NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ29; RAW_FILE_NAME=nRPLC_XZ29 SUBJECT_SAMPLE_FACTORS - XZ3 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ3; RAW_FILE_NAME=nRPLC_XZ3 SUBJECT_SAMPLE_FACTORS - XZ30 Cell type:Quiescent NSC | Age:Old (20-22 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 1 RAW_FILE_NAME=pRPLC_XZ30; RAW_FILE_NAME=nRPLC_XZ30 SUBJECT_SAMPLE_FACTORS - XZ31 Cell type:Activated NSC | Age:Pilot sample, not included in study | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Pilot sample, not included in study RAW_FILE_NAME=pRPLC_XZ31; RAW_FILE_NAME=nRPLC_XZ31 SUBJECT_SAMPLE_FACTORS - XZ32 Cell type:Activated NSC | Age:Pilot sample, not included in study | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Pilot sample, not included in study RAW_FILE_NAME=pRPLC_XZ32; RAW_FILE_NAME=nRPLC_XZ32 SUBJECT_SAMPLE_FACTORS - XZ33 Cell type:Activated NSC | Age:Pilot sample, not included in study | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Pilot sample, not included in study RAW_FILE_NAME=pRPLC_XZ33; RAW_FILE_NAME=nRPLC_XZ33 SUBJECT_SAMPLE_FACTORS - XZ34 Cell type:Activated NSC | Age:Pilot sample, not included in study | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Pilot sample, not included in study RAW_FILE_NAME=pRPLC_XZ34; RAW_FILE_NAME=nRPLC_XZ34 SUBJECT_SAMPLE_FACTORS - XZ4 Cell type:Activated NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ4; RAW_FILE_NAME=nRPLC_XZ4 SUBJECT_SAMPLE_FACTORS - XZ5 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ5; RAW_FILE_NAME=nRPLC_XZ5 SUBJECT_SAMPLE_FACTORS - XZ6 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ6; RAW_FILE_NAME=nRPLC_XZ6 SUBJECT_SAMPLE_FACTORS - XZ7 Cell type:Activated NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ7; RAW_FILE_NAME=nRPLC_XZ7 SUBJECT_SAMPLE_FACTORS - XZ8 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ8; RAW_FILE_NAME=nRPLC_XZ8 SUBJECT_SAMPLE_FACTORS - XZ9 Cell type:Quiescent NSC | Age:Young (3-5 months) | Source:Primary neural stem cell (NSC) culture | Sample type:Whole cell extract | Batch:Batch 2 RAW_FILE_NAME=pRPLC_XZ9; RAW_FILE_NAME=nRPLC_XZ9 #COLLECTION CO:COLLECTION_SUMMARY NSCs were isolated from male C57BL/6 mice as previously described1-3 . Briefly, CO:COLLECTION_SUMMARY subventricular zones (SVZs) from each brain were microdissected and finely CO:COLLECTION_SUMMARY minced. Tissue suspension was then digested for 35min at 37°C with gentle CO:COLLECTION_SUMMARY agitation in HBSS media (Corning, 21-021-CV) containing 2U/ml Papain CO:COLLECTION_SUMMARY (Worthington LS003124), 1U/ml Dispase II (STEMCELL Technologies, 07913), and CO:COLLECTION_SUMMARY 0.1mg/ml DNase I (Sigma, DN25-100mg), and mechanically dissociated. Isolated CO:COLLECTION_SUMMARY cells were expanded as neurospheres in culture in “Proliferative NSC media” CO:COLLECTION_SUMMARY (NeuroBasal-A medium (Gibco, 10888- 022) with penicillin-streptomycin-glutamine CO:COLLECTION_SUMMARY diluted 1X (Gibco, 10378-016), 2% B27 minus vitamin A (Gibco, 12587-010), CO:COLLECTION_SUMMARY 20ng/ml bFGF (Peprotech, 100-18B) and 20ng/ml EGF (Peprotech, AF-100-15)) at CO:COLLECTION_SUMMARY 37°C in 5% CO2 and 20% O2 at 95% humidity. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY To generate parallel cultures of quiescent and activated NSCs (qNSCs and aNSCs, TR:TREATMENT_SUMMARY respectively), we used a previously described protocol4 . Specifically, 1x106 TR:TREATMENT_SUMMARY NSCs (proliferating, passage 3 to passage 5) were plated in each well of a TR:TREATMENT_SUMMARY 6-well plate (80-90% density). To generate primary cultures of quiescent NSCs TR:TREATMENT_SUMMARY (qNSCs), tissue culture plates were pre-treated with PBS (Corning, 21-040-CV) TR:TREATMENT_SUMMARY containing 50ng/mL Poly-D-Lysine (Sigma-Aldrich, P6407) for 2 hours in 37°C TR:TREATMENT_SUMMARY tissue culture incubator, and then washed 3 times with PBS prior to plating TR:TREATMENT_SUMMARY cells. NSCs were then cultured in “Quiescence NSC media” (NeuroBasal-A TR:TREATMENT_SUMMARY (Gibco, 10888-022), penicillinstreptomycin-glutamine 1X (Gibco, 10378-016), 2% TR:TREATMENT_SUMMARY B27 minus vitamin A (Gibco, 12587-010), 20ng/ml bFGF (Peprotech, 100-18B) and TR:TREATMENT_SUMMARY 50ng/ml BMP4 (Biolegend, 595302). For lipidomics analysis, generation of giant TR:TREATMENT_SUMMARY plasma membrane vesicles (GPMVs) and membrane order assay by Laurdan staining, TR:TREATMENT_SUMMARY qNSCs were incubated in this quiescence media for 7 days before the experiment. TR:TREATMENT_SUMMARY For CRISPR/Cas9 knockout, qNSCs were incubated in quiescent media for 4 days TR:TREATMENT_SUMMARY before 2 additional days of lentiviral transduction in the same media. For all TR:TREATMENT_SUMMARY experiments on qNSCs, quiescence media was replaced every 2 days. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For lipidomics, aNSCs and qNSCs were washed twice with PBS before incubating in SP:SAMPLEPREP_SUMMARY “Proliferative NSC media” minus B27 supplement and “Quiescent NSC media” SP:SAMPLEPREP_SUMMARY minus B27 supplement, respectively. Cells were incubated for 3 hours in these SP:SAMPLEPREP_SUMMARY media at 37°C incubator with 5% CO2 and 20% oxygen at 95% humidity to remove SP:SAMPLEPREP_SUMMARY exogenous lipids contained in B27 supplement. At the end of the incubation SP:SAMPLEPREP_SUMMARY period, cells were washed once with PBS 1X (Corning, 21-040-CV) and scraped into SP:SAMPLEPREP_SUMMARY 500μl of ice-cold PBS using cell lifter (Fisher Scientific 07-200- 364). The SP:SAMPLEPREP_SUMMARY cell suspension was collected in 2ml amber glass vials (Thermo Scientific, SP:SAMPLEPREP_SUMMARY 03-FISVA) sealed with polyethylene cap with PTFE/silicone septum (Waters, SP:SAMPLEPREP_SUMMARY 186000274). All samples were immediately snap-frozen in liquid nitrogen and SP:SAMPLEPREP_SUMMARY stored at -80°C. Lipids were extracted from cell suspension after thawing on SP:SAMPLEPREP_SUMMARY ice using a modified Folch method5 . All chemical reagents used were LC-MS grade SP:SAMPLEPREP_SUMMARY unless indicated otherwise. Specifically, 300μl of cold 100% methanol (Fisher SP:SAMPLEPREP_SUMMARY Scientific, A456-500) containing deuterated lipid standards listed below was SP:SAMPLEPREP_SUMMARY added to the cell suspension. Deuterated triacylglycerol TG(17:0-17:1-17:0(d5)) SP:SAMPLEPREP_SUMMARY (Avanti Polar Lipids, 860903) 0.1μg was used for normalization in the SP:SAMPLEPREP_SUMMARY untargeted LC-MS analysis for lipidomics on activated and quiescent primary NSC SP:SAMPLEPREP_SUMMARY culture (primary NSC culture experiment #1). A mixture containing 54 deuterated SP:SAMPLEPREP_SUMMARY standards (SCIEX, 5040156, LPISTDKIT101) was used for the targeted Lipidyzer SP:SAMPLEPREP_SUMMARY analysis (20μl/sample). A mixture containing 13 deuterated standards SP:SAMPLEPREP_SUMMARY (EquiSPLASH® mix, Avanti Polar Lipids, 330731) and deuterated oleic acid SP:SAMPLEPREP_SUMMARY (Cayman Chemical, 9000432) was used for the untargeted LC-MS analysis SP:SAMPLEPREP_SUMMARY (1μl/sample) on quiescent NSC cultures with CRISPR/Cas9 knockout (primary NSC SP:SAMPLEPREP_SUMMARY culture experiment #2). Homogenates were sonicated three times for 30s each time SP:SAMPLEPREP_SUMMARY at room temperature in a water bath sonicator (VWR, 97043-960). Samples were SP:SAMPLEPREP_SUMMARY rested on ice for 30s between each cycle. Following this step, 600μl of cold SP:SAMPLEPREP_SUMMARY chloroform (Acros Organics, AC610281000, stored at -20°C) was added to the SP:SAMPLEPREP_SUMMARY homogenates. Samples were then subjected to vigorous vortex at 4°C for 30min. SP:SAMPLEPREP_SUMMARY Biphasic separation was achieved after centrifugation at 3000rpm for 10min at SP:SAMPLEPREP_SUMMARY 4°C. The lower organic phase containing the lipids was collected and dried down SP:SAMPLEPREP_SUMMARY under a nitrogen stream using a TurboVap Classic LV (Biotage) at a flow rate of SP:SAMPLEPREP_SUMMARY 0.5l/min for 15min until no visible solution remains, and with dried lipid film SP:SAMPLEPREP_SUMMARY formed at the bottom. Dried lipids were then resolubilized in 200μl 100% SP:SAMPLEPREP_SUMMARY methanol at room temperature before moving to -20°C for storage. On the day of SP:SAMPLEPREP_SUMMARY analysis, for untargeted LC-MS/MS, half of each sample’s lipid extract was SP:SAMPLEPREP_SUMMARY dried down under a nitrogen stream and resolubilized in 200μl of SP:SAMPLEPREP_SUMMARY methanol:toluene (90:10, vol:vol) for analysis on complex lipids, and the other SP:SAMPLEPREP_SUMMARY half of the lipid extract was resolubilized in 100μl of 5% acetonitrile for SP:SAMPLEPREP_SUMMARY free fatty acid analysis. For targeted assay on the Lipidyzer platform, samples SP:SAMPLEPREP_SUMMARY were solubilized in 300μl of 10mM ammonium acetate in methanol:toluene (90:10, SP:SAMPLEPREP_SUMMARY vol:vol) before analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Thermo Accucore C18 (2.1 x 150mm, 2.6μm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Lipid extracts were analyzed in a randomized order using an Ultimate 3000 RSLC MS:MS_COMMENTS system coupled with a Q Exactive plus mass spectrometer (Thermo Scientific) as MS:MS_COMMENTS previously described6 . Each sample was run twice in positive and negative MS:MS_COMMENTS ionization modes. Lipids were separated using an Accucore C18 column 2.1 x MS:MS_COMMENTS 150mm, 2.6μm (Thermo Scientific, 17126-152130) and mobile phase solvents MS:MS_COMMENTS consisted of 10mM ammonium acetate and 0.1% formic acid in 60/40 MS:MS_COMMENTS acetonitrile/water (A) and 10mM ammonium acetate and 0.1% formic acid in 90/10 MS:MS_COMMENTS isopropanol/acetonitrile (B). The gradient profile used was 30% B for 3min, MS:MS_COMMENTS 30-43% in 2min, 43-55% B in 0.1min, 55-65% in 10min, 65-85% B in 6min, 85-100% B MS:MS_COMMENTS in 2min and 100% B for 5min. Lipids were eluted from the column at 0.4ml/min, MS:MS_COMMENTS the oven temperature was set at 45°C, and the injection volume was 5μl. MS:MS_COMMENTS Autosampler temperature was set at 20°C to prevent lipid aggregation. The Q MS:MS_COMMENTS Exactive was equipped with a HESI-II probe and operated in data-dependent MS:MS_COMMENTS acquisition mode for whole cell samples with CRISPR/Cas9 knockouts. To increase MS:MS_COMMENTS sensitivity in untreated whole cell and giant plasma membrane vesicles (GPMVs) MS:MS_COMMENTS samples (see below), samples were run in full MS mode and MS/MS spectra were MS:MS_COMMENTS acquired on pooled samples. To maximize the number of identified lipids, the 100 MS:MS_COMMENTS most abundant peaks found in blanks were excluded from MS/MS events. External MS:MS_COMMENTS calibration was performed using an infusion of Pierce LTQ Velos ESI Positive Ion MS:MS_COMMENTS Calibration Solution or Pierce ESI Negative Ion Calibration Solution. Data MS:MS_COMMENTS quality was ensured by 1) injecting 6 pooled samples to equilibrate the LC-MS MS:MS_COMMENTS system prior to run the sequence, 2) checking mass accuracy, retention time, and MS:MS_COMMENTS peak shape of internal standards in each sample. Data from each mode were MS:MS_COMMENTS independently analyzed using Progenesis QI software (v2.3, Nonlinear Dynamics). MS:MS_COMMENTS Metabolic features from blanks and that did not show sufficient linearity upon MS:MS_COMMENTS dilution in QC samples (r < 0.6) were discarded. Only metabolic features present MS:MS_COMMENTS in >2/3 of the samples were kept for further analysis. Lipids were identified MS:MS_COMMENTS using LipidSearch (v4.3, Thermo Scientific) by matching the precursor ion mass MS:MS_COMMENTS to a database and the experimental MS/MS spectra to a spectral library MS:MS_COMMENTS containing theoretical fragmentation spectra. The most abundant ion adduct was MS:MS_COMMENTS selected for each lipid class for downstream analysis and quantification. MS:MS_COMMENTS Specifically, in positive mode, [M (molecular ion) +H]+ for MS:MS_COMMENTS Lysophosphatidylcholine (LPC), Phosphatidylcholine (PC), MS:MS_COMMENTS Phosphatidylethanolamine (PE), Sphingomyelin (SM), Acylcarnitine (AcCa) and MS:MS_COMMENTS Coenzyme (Co), [M+NH4]+ for Cholesterol ester (ChE), Monoacylglycerol (MG), MS:MS_COMMENTS Diacylglycerol (DG) and Triacylglycerol (TG), [M+H-H2O]+ for Ceramide (Cer) and MS:MS_COMMENTS Cholesterol. In negative mode, [M-H]- for Lysophosphatidylethanolamine (LPE), MS:MS_COMMENTS Phosphatidylinositol (PI), Phosphatidylserine (PS), Phosphatidylglycerol (PG), MS:MS_COMMENTS Cardiolipin (CL), Lysophosphatidylinositol (LPI) and Lysophosphatidylserine MS:MS_COMMENTS (LPS). To reduce the risk of misidentification, MS/MS spectra from lipids of MS:MS_COMMENTS interest were manually investigated to validate the assignments. The manual MS:MS_COMMENTS validation consisted in verifying that: 1) both positive and negative mode MS/MS MS:MS_COMMENTS spectra matched the expected fragments, 2) the main lipid adduct forms detected MS:MS_COMMENTS in positive and negative modes were in agreement with the lipid class MS:MS_COMMENTS identified, 3) the retention time was compatible with the lipid class MS:MS_COMMENTS identified, and 4) the peak shape was acceptable. The fragmentation pattern of MS:MS_COMMENTS each lipid class detected was experimentally validated using lipid internal MS:MS_COMMENTS standards. In primary NSC culture experiment #1, internal standard MS:MS_COMMENTS (TG(17:0-17:1-17:0(d5)), see above) - normalized signal intensity was obtained MS:MS_COMMENTS for all detected lipids. Subsequently, median lipid intensity of each sample was MS:MS_COMMENTS used to normalize for variation in starting material before performing MS:MS_COMMENTS downstream analyses. In primary NSC culture experiment #2, the inclusion of MS:MS_COMMENTS EquiSPLASH® deuterated lipid standard mix (see above) allowed us to obtain MS:MS_COMMENTS quantitative molar concentration for identified lipids that belong to the 13 MS:MS_COMMENTS lipid classes (PC, LPC, PE, LPE, PG, PI, PS, TG, DG, MG, ChE, Cer, SM). For MS:MS_COMMENTS those lipids, single-point internal standard calibrations were performed to MS:MS_COMMENTS estimate absolute concentrations for each lipid. A number of less abundant lipid MS:MS_COMMENTS classes of Cholesterol (Chol), Cardiolipin (CL), Acyl carnitine (AcCa), Coenzyme MS:MS_COMMENTS (Co), Sphingosine phosphate (SPHP) and Zymosterol ester (ZyE) were also detected MS:MS_COMMENTS in primary NSC culture experiment #2. As these lipids do not have internal MS:MS_COMMENTS standard for their respective lipid classes, normalized signal intensity was MS:MS_COMMENTS obtained instead of molar concentration. Normalized intensity for each lipid was MS:MS_COMMENTS calculated using the median lipid molar concentration of quantified lipids of MS:MS_COMMENTS each sample to normalize for variation in starting material. Importantly, we MS:MS_COMMENTS ensured linearity within the range of detected endogenous lipids by using serial MS:MS_COMMENTS dilutions of deuterated standards spanning 4 orders of magnitude. Subsequently, MS:MS_COMMENTS median lipid molar concentration of each sample was used to normalize for MS:MS_COMMENTS variation in starting material. MS:MS_RESULTS_FILE ST002257_AN003687_Results.txt UNITS:Spectral count Has m/z:Yes Has RT:Yes RT units:Minutes #END