Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA337398

Local Sample ID:	S06_mitos_linoleic.acid_WT_2
Subject ID:	SU003230
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003230
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
S06_mitos_linoleic.acid_WT_2	SA337398	FL039692	total mitochondria	Sample source
S06_mitos_linoleic.acid_WT_2	SA337398	FL039692	WT	Genotype
S06_mitos_linoleic.acid_WT_2	SA337398	FL039692	linoleic acid	Condition

Collection:

Collection ID:	CO003223
Collection Summary:	Human osteosarcoma U2OS WT, U2OS BAK Ko expressing GFP BAK, and U2OS FADS2 KO cell lines were cultured at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen, Germany). For lipidomic experiments cells were incubated with 1 μM of ABT-737 and S63845 in the complete media and incubated for 50 min at 37°C and 5% CO2. FADS2 KO in U2OS cells was generated in the lab by the CRISPR/Cas9 method. Linoleic acid stock (50 mM) was prepared in ethanol and diluted into culture media before adding them to the cells. Mitochondria were isolated from cultured human osteosarcoma cells by mechanical disruption of cells followed by differential centrifugation: Cells were harvested by trypsinization, washed in PBS, and then resuspend in isolation buffer (IM;250 mM sucrose, 5 mM Tris, and 2 mM EDTA; pH 7.4 and protease inhibitor cocktail) and mechanically broken using glass homogenizer on ice (30-40 strokes on ice) and total cellular lysates were spin down first to remove nuclei and cell debris at 600 x g for 5 min and later at 10,800 x g for 10 min at 4°C to get the crude mitochondria. Mitochondrial pellet was washed 2-3 times with isolation buffer to remove other impurities from mitochondria. Isolated mitochondria were solubilized using SMA co-polymer. For this, mitochondria either from apoptotic or healthy cells were incubated with 0.5% SMA (2:1) for 45 min at room temperature with gentle rotation. Mitochondrial membrane was spun down at 100,000 x g for 40 min to separate solubilized SMALP from the insolubilized membrane. Next, the size of SMALP was analyzed by Dynamic Light Scattering (DLS). For DLS measurements, 15 μl of sample was added to a quartz cuvette which had been thoroughly cleaned with Milli-Q H2O. The cuvette was placed in DynaPro NanoStar (Wyatt Technology corporation, USA) and the sample was analyzed using 10 runs with 10 second acquisition time. This helps to determine the mass distribution of the sample as well as the estimated size of the particles. The distance distribution is shown on a log scale. The size of SMALP as well as the homogeneity with in the sample were also checked by Negative Transmission Electron Microscopy (TEM). For this the diluted SMALPs were placed onto a glow-discharged copper grid (Electron Microscopy Sciences) coated with a layer of thin carbon, washed twice with water, stained with 2% uranyl acetate for 5 min and then air-dried. The grids were imaged on a JEOL JEM2100PLUS electron microscope and recorded with a GATAN OneView camera (CECAD Imaging Facility). mEGFP-BAK-SMALPs were affinity purified from total solubilized mitochondrial membrane fraction (SMALP). For this total SMALP were incubated with 25 μl of GFP-trap MA beads for 90 min with slow rotation in cold room. Beads were washed 2 times with 100 μl of Tris buffer (50 mM Tris 150 mM NaCl pH 8), and finally resuspend in 100 ul of Tris buffer. Small aliquots of unbound and wash fractions were used to analyze the purification quality.
Sample Type:	Mitochondria

Treatment:

Treatment ID:	TR003239
Treatment Summary:	The samples were not subjected to any further treatment.

Sample Preparation:

Sampleprep ID:	SP003237
Sampleprep Summary:	Glycerophospholipids: Lipids from isolated mitochondria treated with or without SMA were extracted using a procedure previously described (Ejsing et al., 2009) with some modifications: 30-100 µl of sample were brought to a volume of 200 µl with 155 mM ammonium carbonate buffer. Lipids were extracted by adding 990 µl of chloroform/methanol 17:1 (v/v) and internal standards (125 pmol PC 17:0-20:4, 138 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 118 pmol PS 17:0-20:4, 61 pmol PG 17:0/20:4, 72 pmol PA 17:0/20:4, 10 µl Cardiolipin Mix I; Avanti Polar Lipids), followed by shaking at 900 rpm/min in a ThermoMixer (Eppendorf) at 20 °C for 30 min. After centrifugation (12,000xg, 5 min, 4 °C), the lower (organic) phase was transferred to a new tube, and the upper phase was extracted again with 990 mL chloroform/methanol 2:1 (v/v). The combined organic phases were dried under a stream of nitrogen. The residues were resolved in 200 µl of methanol. Ceramides and sphingomyelins: For the analysis of ceramides and sphingomyelins in isolated mitochondria without and after SMA treatment, lipids were extracted as described above in the presence of 127 pmol ceramide 12:0 and 124 pmol sphingomyelin 12:0 (internal standards, Avanti Polar Lipids). The dried extracts were resolved in 100 µL of Milli-Q water and 750 µL of chloroform/methanol 1:2 (v/v). Alkaline hydrolysis of glycerolipids was conducted as previously published (Schwamb et al., 2012; Oteng et al., 2017). Fatty acids: To 100 µl of a suspension of isolated mitochondria in PBS, 500 µl of methanol, 250 µl of chloroform, and 0.5 µg palmitic-d31 acid (Sigma-Aldrich) as internal standard were added. The mixture was sonicated for 5 min, and lipids were extracted in a shaking bath at 48 °C for 1 h. Glycerolipids were degraded by alkaline hydrolysis adding 75 µl of 1 M potassium hydroxide in methanol. After 5 min of sonication, the extract was incubated for 1.5 h at 37 °C, and then neutralized with 6 µl of glacial acetic acid. 2 ml of chloroform and 4 ml of water were added to the extract which was vortexed vigorously for 30 sec and then centrifuged (4,000 × g, 5 min, 4 °C) to separate layers. The lower (organic) phase was transferred to a new tube, and the upper phase extracted with additional 2 ml of chloroform. The combined organic phases were dried under a stream of nitrogen. The residues were resolved in 200 µl of acetonitrile/water 2:1 (v/v) and sonicated for 5 min. After centrifugation (12,000 × g, 20 min, 4 °C), 40 µl of the clear supernatants were transferred to autoinjector vials. References: Ejsing et al., Proc Natl Acad Sci USA 2009, 106, 2136; Oteng et al., J Lipid Res 2017, 58, 1100; Schwamb et al., Blood 2012, 120, 3978.

Sampleprep ID:

SP003237

Sampleprep Summary:

Glycerophospholipids: Lipids from isolated mitochondria treated with or without SMA were extracted using a procedure previously described (Ejsing et al., 2009) with some modifications: 30-100 µl of sample were brought to a volume of 200 µl with 155 mM ammonium carbonate buffer. Lipids were extracted by adding 990 µl of chloroform/methanol 17:1 (v/v) and internal standards (125 pmol PC 17:0-20:4, 138 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 118 pmol PS 17:0-20:4, 61 pmol PG 17:0/20:4, 72 pmol PA 17:0/20:4, 10 µl Cardiolipin Mix I; Avanti Polar Lipids), followed by shaking at 900 rpm/min in a ThermoMixer (Eppendorf) at 20 °C for 30 min. After centrifugation (12,000xg, 5 min, 4 °C), the lower (organic) phase was transferred to a new tube, and the upper phase was extracted again with 990 mL chloroform/methanol 2:1 (v/v). The combined organic phases were dried under a stream of nitrogen. The residues were resolved in 200 µl of methanol. Ceramides and sphingomyelins: For the analysis of ceramides and sphingomyelins in isolated mitochondria without and after SMA treatment, lipids were extracted as described above in the presence of 127 pmol ceramide 12:0 and 124 pmol sphingomyelin 12:0 (internal standards, Avanti Polar Lipids). The dried extracts were resolved in 100 µL of Milli-Q water and 750 µL of chloroform/methanol 1:2 (v/v). Alkaline hydrolysis of glycerolipids was conducted as previously published (Schwamb et al., 2012; Oteng et al., 2017). Fatty acids: To 100 µl of a suspension of isolated mitochondria in PBS, 500 µl of methanol, 250 µl of chloroform, and 0.5 µg palmitic-d31 acid (Sigma-Aldrich) as internal standard were added. The mixture was sonicated for 5 min, and lipids were extracted in a shaking bath at 48 °C for 1 h. Glycerolipids were degraded by alkaline hydrolysis adding 75 µl of 1 M potassium hydroxide in methanol. After 5 min of sonication, the extract was incubated for 1.5 h at 37 °C, and then neutralized with 6 µl of glacial acetic acid. 2 ml of chloroform and 4 ml of water were added to the extract which was vortexed vigorously for 30 sec and then centrifuged (4,000 × g, 5 min, 4 °C) to separate layers. The lower (organic) phase was transferred to a new tube, and the upper phase extracted with additional 2 ml of chloroform. The combined organic phases were dried under a stream of nitrogen. The residues were resolved in 200 µl of acetonitrile/water 2:1 (v/v) and sonicated for 5 min. After centrifugation (12,000 × g, 20 min, 4 °C), 40 µl of the clear supernatants were transferred to autoinjector vials. References: Ejsing et al., Proc Natl Acad Sci USA 2009, 106, 2136; Oteng et al., J Lipid Res 2017, 58, 1100; Schwamb et al., Blood 2012, 120, 3978.

Combined analysis:

Analysis ID	AN005102	AN005103	AN005104	AN005105
Analysis type	MS	MS	MS	MS
Chromatography type	None (Direct infusion)	Normal phase	Reversed phase	Reversed phase
Chromatography system	Advion TriVersa NanoMate	Agilent 1260	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	None	Macherey-Nagel Nucleosil NH2 (50×2 mm, 3 um, 120 Å)	Waters Acquity BEH Shield RP18 (100×2.1 mm, 1.7 um)	Phenomenex Core-Shell Kinetex Biphenyl (100×3.0 mm, 2.6 um, 100 Å)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	QTRAP	QTRAP	QTRAP	QTRAP
MS instrument name	SCIEX QTRAP 6500	SCIEX QTRAP 6500	SCIEX QTRAP 6500	SCIEX QTRAP 6500
Ion Mode	POSITIVE	POSITIVE	POSITIVE	NEGATIVE
Units	counts per second (cps)	counts per second (cps)	counts per second (cps)	counts per second (cps)

Chromatography:

Chromatography ID:	CH003858
Chromatography Summary:	Lipid extract infusion and ionization was conducted using Nano-ESI chips with the TriVersa NanoMate operated by the ChipSoft Software (Advion) under the following settings: sample infusion volume: 14 μl, volume of air to aspirate after sample: 1 μl, air gap before chip: enabled, aspiration delay: 0 s, prepiercing: with mandrel, spray sensing: enabled, cooling temperature: 14°C, gas pressure: 0.5 psi, ionization voltage: 1.4 kV, and vent headspace: enabled. Prewetting was done once.
Instrument Name:	Advion TriVersa NanoMate
Column Name:	None
Column Temperature:	N/A
Flow Gradient:	N/A
Flow Rate:	N/A
Solvent A:	None
Solvent B:	None
Chromatography Type:	None (Direct infusion)

Chromatography ID:	CH003859
Chromatography Summary:	Note: Macherey-Nagel does not offer this column as standard, but manufactures it on request.
Instrument Name:	Agilent 1260
Column Name:	Macherey-Nagel Nucleosil NH2 (50×2 mm, 3 um, 120 Å)
Column Temperature:	20
Flow Gradient:	0 min: 0% B, 0.5 min: 0% B, 0.7 min: 10% B, 1.2 min: 10% B, 1.6 min: 18% B, 2.2 min: 18% B, 2.6 min: 100% B, 4.5 min: 100% B, 4.9 min: 0% B, 6.5 min: 0% B
Flow Rate:	0.75 ml/min
Solvent A:	97% acetonitrile/2% methanol/1% acetic acid; 5 mM ammonium acetate
Solvent B:	99% methanol/1% acetic acid; 5 mM ammonium acetate
Chromatography Type:	Normal phase

Chromatography ID:	CH003860
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity BEH Shield RP18 (100×2.1 mm, 1.7 um)
Column Temperature:	50
Flow Gradient:	0 min: 30% B, 0.5 min: 30% B, 4.5 min: 68% B, 20.5 min: 75% B, 21 min: 97% B, 24 min: 97% B, 24.5 min: 30% B, 28 min: 30% B
Flow Rate:	0.4 ml/min
Solvent A:	60% acetonitrile/40% water; 10 mM ammonium formate
Solvent B:	90% isopropanol/10% acetonitrile; 10 mM ammonium formate
Chromatography Type:	Reversed phase

Chromatography ID:	CH003861
Instrument Name:	Shimadzu Nexera X2
Column Name:	Phenomenex Core-Shell Kinetex Biphenyl (100×3.0 mm, 2.6 um, 100 Å)
Column Temperature:	40
Flow Gradient:	0 min: 55% B, 4 min: 95% B, 7 min: 95% B, 7.1 min: 55% B, 10 min: 55% B
Flow Rate:	0.7 ml/min
Solvent A:	100% water; 0.012 % acetic acid; 5 mM ammonium acetate
Solvent B:	80% acetonitrile/20% isopropanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004839
Analysis ID:	AN005102
Instrument Name:	SCIEX QTRAP 6500
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	PC, PE, PI, PS, PG, and PA species were analyzed by Nano-Electrospray Ionization Tandem Spectrometry (Nano-ESI-MS/MS) with direct infusion of the lipid extract (Shotgun Lipidomics) as previously described (Kumar et al., J Cell Biol 2015, 211, 1057).
Ion Mode:	POSITIVE

MS ID:	MS004840
Analysis ID:	AN005103
Instrument Name:	SCIEX QTRAP 6500
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	LC-ESI-MS/MS analysis of ceramides and sphingomyelins was conducted as previously published (Oteng et al., J Lipid Res 2017, 58, 1100; Schwamb et al., Blood 2012, 120, 3978).
Ion Mode:	POSITIVE

MS ID:	MS004841
Analysis ID:	AN005104
Instrument Name:	SCIEX QTRAP 6500
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	Cardiolipin (CL) species were analyzed by Liquid Chromatography coupled to Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). CL species were monitored in the positive ion mode using the following Multiple Reaction Monitoring (MRM) transitions: CL 68:4, m/z 1418.9 to 575.4; CL 70:4, m/z 1446.9 to 575.4; CL 72:4 m/z 1475.0 to 603.4; CL 61:1 (internal standard), m/z 1326.9 to 535.4. For all MRM transitions the values for declustering potential, entrance potential, collision energy, and cell exit potential were 140 V, 10 V, 45 V, and 7 V, respectively (Tatsuta, Methods Mol Biol 2017, 1567, 79). The instrument settings for nebulizer gas (Gas 1), turbo gas (Gas 2), curtain gas, and collision gas were 50 psi, 50 psi, 40 psi, and medium, respectively. The Turbo V ESI source temperature was 500 °C, and the ionspray voltage was 4.5 kV. The LC chromatogram peaks of the endogenous CL species and the internal standard CL 61:1 were integrated using the MultiQuant 3.0.2 software (SCIEX).
Ion Mode:	POSITIVE

MS ID:	MS004842
Analysis ID:	AN005105
Instrument Name:	SCIEX QTRAP 6500
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	Fatty acid levels were determined by LC-ESI-MS/MS: Fatty acids were monitored in the negative ion mode using “pseudo” Multiple Reaction Monitoring (MRM) transitions (Hellmuth et al., Anal Chem 2012, 84, 1483). The instrument settings for nebulizer gas (Gas 1), turbo gas (Gas 2), curtain gas, and collision gas were 60 psi, 90 psi, 40 psi, and medium, respectively. The Turbo V ESI source temperature was 650 °C, and the ionspray voltage was -4 kV. The LC chromatogram peaks of the endogenous fatty acids and the internal standard palmitic-d31 acid were integrated using the MultiQuant 3.0.2 software (SCIEX).
Ion Mode:	NEGATIVE