Summary of Study ST002259

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 PR001444. The data can be accessed directly via it's Project DOI: 10.21228/M82Q5N This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST002259
Study Title	Lipidomic profiling reveals age-dependent changes in complex plasma membrane lipids that regulate neural stem cell aging (Part 3)
Study Summary	The aging brain exhibits a decline in the regenerative populations of neural stem cells (NSCs), which may underlie age-associated defects in sensory and cognitive functions1-4 . While mechanisms that restore old NSC function have started to be identified5-9 , the role of lipids – especially complex lipids – in NSC aging remains largely unclear. Using lipidomic profiling by mass spectrometry, we identify age-related lipidomic signatures in young and old quiescent NSCs in vitro and in vivo. These analyses reveal drastic changes in several complex membrane lipid classes, including phospholipids and sphingolipids in old NSCs. Moreover, polyunsaturated fatty acids (PUFAs) strikingly increase across complex lipid classes in quiescent NSCs during aging. Lipidomic profiling of isolated plasma membrane vesicles shows that agerelated differences in complex lipid levels and side chain composition are largely occurring in plasma membrane lipids. Experimentally, we find that aging is accompanied by modifications in membrane biophysical properties, with a decrease in plasma membrane order in old quiescent NSCs in vitro and in vivo. To determine the functional role of plasma membrane lipids in aging NSCs, we perform genetic and supplementations studies. Knockout of Mboat2, which encodes a phospholipid acyltransferase, exacerbates age-related lipidomic changes in old quiescent NSCs and impedes their ability to activate. As Mboat2 expression declines with age, Mboat2 deficiency may drive NSC decline during aging. Interestingly, supplementation of plasma membrane lipids derived from young NSCs boosts the ability of old quiescent NSCs to activate. Our work could lead to lipid-based strategies for restoring the regenerative potential of NSCs in old individuals, which has important implications to counter brain decline during aging.
Institute	Stanford University
Last Name	Contrepois
First Name	Kevin
Address	300 Pasteur Dr 94305 Stanford
Email	kcontrep@stanford.edu
Phone	650-664-7325
Submit Date	2022-08-15
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-08-12
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001444
Project DOI:	doi: 10.21228/M82Q5N
Project Title:	Lipidomic profiling reveals age-dependent changes in complex plasma membrane lipids that regulate neural stem cell aging
Project Summary:	Study of lipid changes with age in NSC
Institute:	Stanford University
Last Name:	Contrepois
First Name:	Kevin
Address:	300 Pasteur Dr 94305 Stanford
Email:	kcontrep@stanford.edu
Phone:	650-664-7325

Subject:

Subject ID:	SU002345
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammals

Factors:

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

mb_sample_id	local_sample_id	Cell type	Age	Source	Sample type
SA217246	XY_11	Quiescent NSC	Old (20-22 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217247	XY_10	Quiescent NSC	Old (20-22 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217248	XY_5	Quiescent NSC	Old (20-22 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217249	XY_4	Quiescent NSC	Old (20-22 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217250	XY_2	Quiescent NSC	Old (20-22 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217251	XY_9	Quiescent NSC	Old (20-22 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217252	XY_12	Quiescent NSC	Young (3-5 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217253	XY_7	Quiescent NSC	Young (3-5 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217254	XY_3	Quiescent NSC	Young (3-5 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217255	XY_6	Quiescent NSC	Young (3-5 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217256	XY_1	Quiescent NSC	Young (3-5 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract
SA217257	XY_8	Quiescent NSC	Young (3-5 months)	In vivo isolated quiescent neural stem cells (qNSCs)	Whole cell extract

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO002338
Collection Summary:	NSCs were isolated from male C57BL/6 mice as previously described1-3 . Briefly, subventricular zones (SVZs) from each brain were microdissected and finely minced. Tissue suspension was then digested for 35min at 37°C with gentle agitation in HBSS media (Corning, 21-021-CV) containing 2U/ml Papain (Worthington LS003124), 1U/ml Dispase II (STEMCELL Technologies, 07913), and 0.1mg/ml DNase I (Sigma, DN25-100mg), and mechanically dissociated. Isolated cells were expanded as neurospheres in culture in “Proliferative NSC media” (NeuroBasal-A medium (Gibco, 10888- 022) with penicillin-streptomycin-glutamine diluted 1X (Gibco, 10378-016), 2% B27 minus vitamin A (Gibco, 12587-010), 20ng/ml bFGF (Peprotech, 100-18B) and 20ng/ml EGF (Peprotech, AF-100-15)) at 37°C in 5% CO2 and 20% O2 at 95% humidity.
Sample Type:	Brain

Treatment:

Treatment ID:	TR002357
Treatment Summary:	To generate parallel cultures of quiescent and activated NSCs (qNSCs and aNSCs, respectively), we used a previously described protocol4 . Specifically, 1x106 NSCs (proliferating, passage 3 to passage 5) were plated in each well of a 6-well plate (80-90% density). To generate primary cultures of quiescent NSCs (qNSCs), tissue culture plates were pre-treated with PBS (Corning, 21-040-CV) containing 50ng/mL Poly-D-Lysine (Sigma-Aldrich, P6407) for 2 hours in 37°C tissue culture incubator, and then washed 3 times with PBS prior to plating cells. NSCs were then cultured in “Quiescence NSC media” (NeuroBasal-A (Gibco, 10888-022), penicillinstreptomycin-glutamine 1X (Gibco, 10378-016), 2% B27 minus vitamin A (Gibco, 12587-010), 20ng/ml bFGF (Peprotech, 100-18B) and 50ng/ml BMP4 (Biolegend, 595302). For lipidomics analysis, generation of giant plasma membrane vesicles (GPMVs) and membrane order assay by Laurdan staining, qNSCs were incubated in this quiescence media for 7 days before the experiment. For CRISPR/Cas9 knockout, qNSCs were incubated in quiescent media for 4 days before 2 additional days of lentiviral transduction in the same media. For all experiments on qNSCs, quiescence media was replaced every 2 days.

Treatment ID:

TR002357

Treatment Summary:

To generate parallel cultures of quiescent and activated NSCs (qNSCs and aNSCs, respectively), we used a previously described protocol4 . Specifically, 1x106 NSCs (proliferating, passage 3 to passage 5) were plated in each well of a 6-well plate (80-90% density). To generate primary cultures of quiescent NSCs (qNSCs), tissue culture plates were pre-treated with PBS (Corning, 21-040-CV) containing 50ng/mL Poly-D-Lysine (Sigma-Aldrich, P6407) for 2 hours in 37°C tissue culture incubator, and then washed 3 times with PBS prior to plating cells. NSCs were then cultured in “Quiescence NSC media” (NeuroBasal-A (Gibco, 10888-022), penicillinstreptomycin-glutamine 1X (Gibco, 10378-016), 2% B27 minus vitamin A (Gibco, 12587-010), 20ng/ml bFGF (Peprotech, 100-18B) and 50ng/ml BMP4 (Biolegend, 595302). For lipidomics analysis, generation of giant plasma membrane vesicles (GPMVs) and membrane order assay by Laurdan staining, qNSCs were incubated in this quiescence media for 7 days before the experiment. For CRISPR/Cas9 knockout, qNSCs were incubated in quiescent media for 4 days before 2 additional days of lentiviral transduction in the same media. For all experiments on qNSCs, quiescence media was replaced every 2 days.

Sample Preparation:

Sampleprep ID:	SP002351
Sampleprep Summary:	For lipidomics, aNSCs and qNSCs were washed twice with PBS before incubating in “Proliferative NSC media” minus B27 supplement and “Quiescent NSC media” minus B27 supplement, respectively. Cells were incubated for 3 hours in these media at 37°C incubator with 5% CO2 and 20% oxygen at 95% humidity to remove exogenous lipids contained in B27 supplement. At the end of the incubation period, cells were washed once with PBS 1X (Corning, 21-040-CV) and scraped into 500μl of ice-cold PBS using cell lifter (Fisher Scientific 07-200- 364). The cell suspension was collected in 2ml amber glass vials (Thermo Scientific, 03-FISVA) sealed with polyethylene cap with PTFE/silicone septum (Waters, 186000274). All samples were immediately snap-frozen in liquid nitrogen and stored at -80°C. Lipids were extracted from cell suspension after thawing on ice using a modified Folch method5 . All chemical reagents used were LC-MS grade unless indicated otherwise. Specifically, 300μl of cold 100% methanol (Fisher Scientific, A456-500) containing deuterated lipid standards listed below was added to the cell suspension. Deuterated triacylglycerol TG(17:0-17:1-17:0(d5)) (Avanti Polar Lipids, 860903) 0.1μg was used for normalization in the untargeted LC-MS analysis for lipidomics on activated and quiescent primary NSC culture (primary NSC culture experiment #1). A mixture containing 54 deuterated standards (SCIEX, 5040156, LPISTDKIT101) was used for the targeted Lipidyzer analysis (20μl/sample). A mixture containing 13 deuterated standards (EquiSPLASH® mix, Avanti Polar Lipids, 330731) and deuterated oleic acid (Cayman Chemical, 9000432) was used for the untargeted LC-MS analysis (1μl/sample) on quiescent NSC cultures with CRISPR/Cas9 knockout (primary NSC culture experiment #2). Homogenates were sonicated three times for 30s each time at room temperature in a water bath sonicator (VWR, 97043-960). Samples were rested on ice for 30s between each cycle. Following this step, 600μl of cold chloroform (Acros Organics, AC610281000, stored at -20°C) was added to the homogenates. Samples were then subjected to vigorous vortex at 4°C for 30min. Biphasic separation was achieved after centrifugation at 3000rpm for 10min at 4°C. The lower organic phase containing the lipids was collected and dried down under a nitrogen stream using a TurboVap Classic LV (Biotage) at a flow rate of 0.5l/min for 15min until no visible solution remains, and with dried lipid film formed at the bottom. Dried lipids were then resolubilized in 200μl 100% methanol at room temperature before moving to -20°C for storage. On the day of analysis, for untargeted LC-MS/MS, half of each sample’s lipid extract was dried down under a nitrogen stream and resolubilized in 200μl of methanol:toluene (90:10, vol:vol) for analysis on complex lipids, and the other half of the lipid extract was resolubilized in 100μl of 5% acetonitrile for free fatty acid analysis. For targeted assay on the Lipidyzer platform, samples were solubilized in 300μl of 10mM ammonium acetate in methanol:toluene (90:10, vol:vol) before analysis.

Sampleprep ID:

SP002351

Sampleprep Summary:

For lipidomics, aNSCs and qNSCs were washed twice with PBS before incubating in “Proliferative NSC media” minus B27 supplement and “Quiescent NSC media” minus B27 supplement, respectively. Cells were incubated for 3 hours in these media at 37°C incubator with 5% CO2 and 20% oxygen at 95% humidity to remove exogenous lipids contained in B27 supplement. At the end of the incubation period, cells were washed once with PBS 1X (Corning, 21-040-CV) and scraped into 500μl of ice-cold PBS using cell lifter (Fisher Scientific 07-200- 364). The cell suspension was collected in 2ml amber glass vials (Thermo Scientific, 03-FISVA) sealed with polyethylene cap with PTFE/silicone septum (Waters, 186000274). All samples were immediately snap-frozen in liquid nitrogen and stored at -80°C. Lipids were extracted from cell suspension after thawing on ice using a modified Folch method5 . All chemical reagents used were LC-MS grade unless indicated otherwise. Specifically, 300μl of cold 100% methanol (Fisher Scientific, A456-500) containing deuterated lipid standards listed below was added to the cell suspension. Deuterated triacylglycerol TG(17:0-17:1-17:0(d5)) (Avanti Polar Lipids, 860903) 0.1μg was used for normalization in the untargeted LC-MS analysis for lipidomics on activated and quiescent primary NSC culture (primary NSC culture experiment #1). A mixture containing 54 deuterated standards (SCIEX, 5040156, LPISTDKIT101) was used for the targeted Lipidyzer analysis (20μl/sample). A mixture containing 13 deuterated standards (EquiSPLASH® mix, Avanti Polar Lipids, 330731) and deuterated oleic acid (Cayman Chemical, 9000432) was used for the untargeted LC-MS analysis (1μl/sample) on quiescent NSC cultures with CRISPR/Cas9 knockout (primary NSC culture experiment #2). Homogenates were sonicated three times for 30s each time at room temperature in a water bath sonicator (VWR, 97043-960). Samples were rested on ice for 30s between each cycle. Following this step, 600μl of cold chloroform (Acros Organics, AC610281000, stored at -20°C) was added to the homogenates. Samples were then subjected to vigorous vortex at 4°C for 30min. Biphasic separation was achieved after centrifugation at 3000rpm for 10min at 4°C. The lower organic phase containing the lipids was collected and dried down under a nitrogen stream using a TurboVap Classic LV (Biotage) at a flow rate of 0.5l/min for 15min until no visible solution remains, and with dried lipid film formed at the bottom. Dried lipids were then resolubilized in 200μl 100% methanol at room temperature before moving to -20°C for storage. On the day of analysis, for untargeted LC-MS/MS, half of each sample’s lipid extract was dried down under a nitrogen stream and resolubilized in 200μl of methanol:toluene (90:10, vol:vol) for analysis on complex lipids, and the other half of the lipid extract was resolubilized in 100μl of 5% acetonitrile for free fatty acid analysis. For targeted assay on the Lipidyzer platform, samples were solubilized in 300μl of 10mM ammonium acetate in methanol:toluene (90:10, vol:vol) before analysis.

Combined analysis:

Analysis ID	AN003690	AN003691
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	Thermo Accucore C18 (150 x 2.1mm,2.6m)	Thermo Accucore C18 (150 x 2.1mm,2.6m)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Spectral count	Spectral count

Chromatography:

Chromatography ID:	CH002735
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Thermo Accucore C18 (150 x 2.1mm,2.6m)
Column Temperature:	45
Flow Gradient:	30% B for 3min, 30-43% in 2min, 43-55% B in 0.1min, 55-65% in 10min, 65-85% B in 6min, 85-100% B in 2min and 100% B for 5min.
Flow Rate:	0.4ml/min
Solvent A:	60% acetonitrile/40% water; 0.1% formic acid; 10mM ammonium acetate
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003441
Analysis ID:	AN003690
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Lipid extracts were analyzed in a randomized order using an Ultimate 3000 RSLC system coupled with a Q Exactive plus mass spectrometer (Thermo Scientific) as previously described6 . Each sample was run twice in positive and negative ionization modes. Lipids were separated using an Accucore C18 column 2.1 x 150mm, 2.6μm (Thermo Scientific, 17126-152130) and mobile phase solvents consisted of 10mM ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10mM ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The gradient profile used was 30% B for 3min, 30-43% in 2min, 43-55% B in 0.1min, 55-65% in 10min, 65-85% B in 6min, 85-100% B in 2min and 100% B for 5min. Lipids were eluted from the column at 0.4ml/min, the oven temperature was set at 45°C, and the injection volume was 5μl. Autosampler temperature was set at 20°C to prevent lipid aggregation. The Q Exactive was equipped with a HESI-II probe and operated in data-dependent acquisition mode for whole cell samples with CRISPR/Cas9 knockouts. To increase sensitivity in untreated whole cell and giant plasma membrane vesicles (GPMVs) samples (see below), samples were run in full MS mode and MS/MS spectra were acquired on pooled samples. To maximize the number of identified lipids, the 100 most abundant peaks found in blanks were excluded from MS/MS events. External calibration was performed using an infusion of Pierce LTQ Velos ESI Positive Ion Calibration Solution or Pierce ESI Negative Ion Calibration Solution. Data quality was ensured by 1) injecting 6 pooled samples to equilibrate the LC-MS system prior to run the sequence, 2) checking mass accuracy, retention time, and peak shape of internal standards in each sample. Data from each mode were independently analyzed using Progenesis QI software (v2.3, Nonlinear Dynamics). Metabolic features from blanks and that did not show sufficient linearity upon dilution in QC samples (r < 0.6) were discarded. Only metabolic features present in >2/3 of the samples were kept for further analysis. Lipids were identified using LipidSearch (v4.3, Thermo Scientific) by matching the precursor ion mass to a database and the experimental MS/MS spectra to a spectral library containing theoretical fragmentation spectra. The most abundant ion adduct was selected for each lipid class for downstream analysis and quantification. Specifically, in positive mode, [M (molecular ion) +H]+ for Lysophosphatidylcholine (LPC), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Sphingomyelin (SM), Acylcarnitine (AcCa) and Coenzyme (Co), [M+NH4]+ for Cholesterol ester (ChE), Monoacylglycerol (MG), Diacylglycerol (DG) and Triacylglycerol (TG), [M+H-H2O]+ for Ceramide (Cer) and Cholesterol. In negative mode, [M-H]- for Lysophosphatidylethanolamine (LPE), Phosphatidylinositol (PI), Phosphatidylserine (PS), Phosphatidylglycerol (PG), Cardiolipin (CL), Lysophosphatidylinositol (LPI) and Lysophosphatidylserine (LPS). To reduce the risk of misidentification, MS/MS spectra from lipids of interest were manually investigated to validate the assignments. The manual validation consisted in verifying that: 1) both positive and negative mode MS/MS spectra matched the expected fragments, 2) the main lipid adduct forms detected in positive and negative modes were in agreement with the lipid class identified, 3) the retention time was compatible with the lipid class identified, and 4) the peak shape was acceptable. The fragmentation pattern of each lipid class detected was experimentally validated using lipid internal standards. In primary NSC culture experiment #1, internal standard (TG(17:0-17:1-17:0(d5)), see above) - normalized signal intensity was obtained for all detected lipids. Subsequently, median lipid intensity of each sample was used to normalize for variation in starting material before performing downstream analyses. In primary NSC culture experiment #2, the inclusion of EquiSPLASH® deuterated lipid standard mix (see above) allowed us to obtain quantitative molar concentration for identified lipids that belong to the 13 lipid classes (PC, LPC, PE, LPE, PG, PI, PS, TG, DG, MG, ChE, Cer, SM). For those lipids, single-point internal standard calibrations were performed to estimate absolute concentrations for each lipid. A number of less abundant lipid classes of Cholesterol (Chol), Cardiolipin (CL), Acyl carnitine (AcCa), Coenzyme (Co), Sphingosine phosphate (SPHP) and Zymosterol ester (ZyE) were also detected in primary NSC culture experiment #2. As these lipids do not have internal standard for their respective lipid classes, normalized signal intensity was obtained instead of molar concentration. Normalized intensity for each lipid was calculated using the median lipid molar concentration of quantified lipids of each sample to normalize for variation in starting material. Importantly, we ensured linearity within the range of detected endogenous lipids by using serial dilutions of deuterated standards spanning 4 orders of magnitude. Subsequently, median lipid molar concentration of each sample was used to normalize for variation in starting material.
Ion Mode:	POSITIVE

MS ID:	MS003442
Analysis ID:	AN003691
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Lipid extracts were analyzed in a randomized order using an Ultimate 3000 RSLC system coupled with a Q Exactive plus mass spectrometer (Thermo Scientific) as previously described6 . Each sample was run twice in positive and negative ionization modes. Lipids were separated using an Accucore C18 column 2.1 x 150mm, 2.6μm (Thermo Scientific, 17126-152130) and mobile phase solvents consisted of 10mM ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10mM ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The gradient profile used was 30% B for 3min, 30-43% in 2min, 43-55% B in 0.1min, 55-65% in 10min, 65-85% B in 6min, 85-100% B in 2min and 100% B for 5min. Lipids were eluted from the column at 0.4ml/min, the oven temperature was set at 45°C, and the injection volume was 5μl. Autosampler temperature was set at 20°C to prevent lipid aggregation. The Q Exactive was equipped with a HESI-II probe and operated in data-dependent acquisition mode for whole cell samples with CRISPR/Cas9 knockouts. To increase sensitivity in untreated whole cell and giant plasma membrane vesicles (GPMVs) samples (see below), samples were run in full MS mode and MS/MS spectra were acquired on pooled samples. To maximize the number of identified lipids, the 100 most abundant peaks found in blanks were excluded from MS/MS events. External calibration was performed using an infusion of Pierce LTQ Velos ESI Positive Ion Calibration Solution or Pierce ESI Negative Ion Calibration Solution. Data quality was ensured by 1) injecting 6 pooled samples to equilibrate the LC-MS system prior to run the sequence, 2) checking mass accuracy, retention time, and peak shape of internal standards in each sample. Data from each mode were independently analyzed using Progenesis QI software (v2.3, Nonlinear Dynamics). Metabolic features from blanks and that did not show sufficient linearity upon dilution in QC samples (r < 0.6) were discarded. Only metabolic features present in >2/3 of the samples were kept for further analysis. Lipids were identified using LipidSearch (v4.3, Thermo Scientific) by matching the precursor ion mass to a database and the experimental MS/MS spectra to a spectral library containing theoretical fragmentation spectra. The most abundant ion adduct was selected for each lipid class for downstream analysis and quantification. Specifically, in positive mode, [M (molecular ion) +H]+ for Lysophosphatidylcholine (LPC), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Sphingomyelin (SM), Acylcarnitine (AcCa) and Coenzyme (Co), [M+NH4]+ for Cholesterol ester (ChE), Monoacylglycerol (MG), Diacylglycerol (DG) and Triacylglycerol (TG), [M+H-H2O]+ for Ceramide (Cer) and Cholesterol. In negative mode, [M-H]- for Lysophosphatidylethanolamine (LPE), Phosphatidylinositol (PI), Phosphatidylserine (PS), Phosphatidylglycerol (PG), Cardiolipin (CL), Lysophosphatidylinositol (LPI) and Lysophosphatidylserine (LPS). To reduce the risk of misidentification, MS/MS spectra from lipids of interest were manually investigated to validate the assignments. The manual validation consisted in verifying that: 1) both positive and negative mode MS/MS spectra matched the expected fragments, 2) the main lipid adduct forms detected in positive and negative modes were in agreement with the lipid class identified, 3) the retention time was compatible with the lipid class identified, and 4) the peak shape was acceptable. The fragmentation pattern of each lipid class detected was experimentally validated using lipid internal standards. In primary NSC culture experiment #1, internal standard (TG(17:0-17:1-17:0(d5)), see above) - normalized signal intensity was obtained for all detected lipids. Subsequently, median lipid intensity of each sample was used to normalize for variation in starting material before performing downstream analyses. In primary NSC culture experiment #2, the inclusion of EquiSPLASH® deuterated lipid standard mix (see above) allowed us to obtain quantitative molar concentration for identified lipids that belong to the 13 lipid classes (PC, LPC, PE, LPE, PG, PI, PS, TG, DG, MG, ChE, Cer, SM). For those lipids, single-point internal standard calibrations were performed to estimate absolute concentrations for each lipid. A number of less abundant lipid classes of Cholesterol (Chol), Cardiolipin (CL), Acyl carnitine (AcCa), Coenzyme (Co), Sphingosine phosphate (SPHP) and Zymosterol ester (ZyE) were also detected in primary NSC culture experiment #2. As these lipids do not have internal standard for their respective lipid classes, normalized signal intensity was obtained instead of molar concentration. Normalized intensity for each lipid was calculated using the median lipid molar concentration of quantified lipids of each sample to normalize for variation in starting material. Importantly, we ensured linearity within the range of detected endogenous lipids by using serial dilutions of deuterated standards spanning 4 orders of magnitude. Subsequently, median lipid molar concentration of each sample was used to normalize for variation in starting material.
Ion Mode:	NEGATIVE