Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA337379

Local Sample ID:	S05_pulldown_Control_31.09
Subject ID:	SU003230
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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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.