Summary of Study ST003114

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 PR001935. The data can be accessed directly via it's Project DOI: 10.21228/M8M430 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.

Study ID	ST003114
Study Title	Lipidomics analyses in model membranes, isolated mitochondria and cellular systems to study how the local lipid environment affects BAX and BAK function during apoptosis.
Study Summary	To investigate how the local lipid environment affects BAX and BAK function during apoptosis, we performed quantitative analyses of different lipid classes (glycerophospholipids, fatty acids, ceramides and sphingomyelins) in cultured cells, isolated mitochondria and lipid nanodics formed by Styrene-Malic Acid (SMA) co-polymers. Ceramides, sphingomyelins, fatty acids and cardiolipins were analyzed by Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS). For glycerophospholipids (PC, PE, PI, PS, PG, PA) we applied direct infusion MS approaches (Shotgun Lipidomics).
Institute	University of Cologne
Department	Faculty of Medicine and University Hospital of Cologne, Cluster of Excellence Cellular Stress Responses in Aging-associated Diseases (CECAD)
Laboratory	CECAD Lipidomics/Metabolomics Facility
Last Name	Brodesser
First Name	Susanne
Address	Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany
Email	susanne.brodesser@uk-koeln.de
Phone	+49 221 478 84015
Submit Date	2023-08-16
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-03-13
Release Version	1

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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.