Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA237007

Local Sample ID:	S03_B.cells_tfgWT_4A1
Subject ID:	SU002449
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002449
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
S03_B.cells_tfgWT_4A1	SA237007	FL029343	tfgWT	Genotype

Collection:

Collection ID:	CO002442
Collection Summary:	CH12tfgWT and CH12tfgKO B cells (Steinmetz et al. 2021, Autophagy 17(9):2238-2256) were cultured in R10 medium (RPMI1640 [Gibco Life Technologies, 31870-025], 10% FCS, 2 mM glutamate [Gibco Life Technologies, 25030-024], 1 mM sodium pyruvate [Gibco Life Technologies, 11360-039], 50 U/ml penicillin G, 50 μg/ml streptomycin [Gibco Life Technologies, 15140-122], 50 μM β-mercaptoethanol [Gibco Life Technologies, 31350-010]) at 37°C and 5% CO2.
Sample Type:	CH12 B lymphoma cells

Treatment:

Treatment ID:	TR002461
Treatment Summary:	The cells have not undergone any further treatment.

Sample Preparation:

Sampleprep ID:	SP002455
Sampleprep Summary:	Glycerophospholipids (PC, PE, PI, PS, PG, PA) in B cells were analyzed by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI-MS/MS) with direct infusion of the lipid extract (Shotgun Lipidomics): 14 to 45 million cells were homogenized in 300 µl of Milli-Q water using the Precellys 24 Homogenisator (Peqlab, Erlangen, Germany) at 6.500 rpm for 30 sec. The protein content of the homogenate was routinely determined using bicinchoninic acid. To 100 µl of the homogenate 400 µl of Milli-Q water, 1.875 ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PC 17:0-20:4, 132 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 131 pmol PS 17:0-20:4, 62 pmol PG 17:0/20:4, 75 pmol PA 17:0/20:4; Avanti Polar Lipids) were added. Lipids were extracted using the “One-Step Extraction” as described in Özbalci et al. 2013, Methods Mol Biol 1033:3-20. Dried lipid extracts were dissolved in 300 μl of methanol. 20 μl of the lipid extract in methanol were loaded into 96-well plates and diluted with 20 μl of 20 mM ammonium acetate in methanol.

Sampleprep ID:

SP002455

Sampleprep Summary:

Glycerophospholipids (PC, PE, PI, PS, PG, PA) in B cells were analyzed by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI-MS/MS) with direct infusion of the lipid extract (Shotgun Lipidomics): 14 to 45 million cells were homogenized in 300 µl of Milli-Q water using the Precellys 24 Homogenisator (Peqlab, Erlangen, Germany) at 6.500 rpm for 30 sec. The protein content of the homogenate was routinely determined using bicinchoninic acid. To 100 µl of the homogenate 400 µl of Milli-Q water, 1.875 ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PC 17:0-20:4, 132 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 131 pmol PS 17:0-20:4, 62 pmol PG 17:0/20:4, 75 pmol PA 17:0/20:4; Avanti Polar Lipids) were added. Lipids were extracted using the “One-Step Extraction” as described in Özbalci et al. 2013, Methods Mol Biol 1033:3-20. Dried lipid extracts were dissolved in 300 μl of methanol. 20 μl of the lipid extract in methanol were loaded into 96-well plates and diluted with 20 μl of 20 mM ammonium acetate in methanol.

Combined analysis:

Analysis ID	AN003854
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	none
Column	none
MS Type	ESI
MS instrument type	QTRAP
MS instrument name	SCIEX QTRAP 6500
Ion Mode	POSITIVE
Units	counts per second (cps)

Chromatography:

Chromatography ID:	CH002852
Chromatography Summary:	Lipid 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:	none
Column Name:	none
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS003595
Analysis ID:	AN003854
Instrument Name:	SCIEX QTRAP 6500
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	Mass spectrometric analysis was performed using the QTRAP 6500 (SCIEX) operated by Analyst 1.6.3. The following instrument dependent settings were used: curtain gas, 20 psi; CAD gas, medium; and interface heater temperature, 100°C. PC analysis was performed in the positive ion mode by scanning for precursors of m/z 184 at a collision energy of 35 eV. PE, PS, PG, PI, and PA measurements were performed in the positive ion mode by scanning for neutral losses of 141, 185, 189, 277, and 115 D at CE of 25 eV. The value for the declustering potential was 100 V (Özbalci et al. 2013, Methods Mol Biol 1033:3-20). Scanning was performed in a mass range of m/z 650–900 D and at a scan rate of 200 D/s. 61 MCA spectra were accumulated. Mass spectra were processed by the LipidView Software Version 1.2 (SCIEX) for identification and quantification of glycerophospholipids. Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards.
Ion Mode:	POSITIVE