Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA235414

Local Sample ID:	FContB-4-PE
Subject ID:	SU002435
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606/10090

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Subject:

Subject ID:	SU002435
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606/10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
FContB-4-PE	SA235414	FL029199	Control2	Genotype

Collection:

Collection ID:	CO002428
Collection Summary:	Fibroblasts and MEFs were cultured under 5% CO2 at 37°C in Dulbecco's modified medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. For the extraction of phospholipids, fibroblasts were seeded in 6-well plate and cultured overnight, followed by replacement of fresh culture medium. Cells were harvested at 48 hours after the medium change as described below.
Sample Type:	Cultured fibroblasts

Treatment:

Treatment ID:	TR002447
Treatment Summary:	No treatment

Sample Preparation:

Sampleprep ID:	SP002441
Sampleprep Summary:	Cells were detached by incubation with trypsin and suspended in PBS. Protein concentration was determined by the bicinchonic acid method (Thermo Fisher Scientific, Rockford, IL). Total lipids were extracted from 50 μg of cellular protein by the Bligh and Dyer method. Cells were dissolved in methanol/chloroform/water at 2:1:0.8 v/v/v and then 50 pmol of 1,2-adidodecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Alabaster, AL) and 1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids) were added as internal standards. After incubation for 5 min at room temperature, 1 ml each of water and chloroform were added and the samples were then centrifuged at 2,000 rpm for 5 min in Himac CF-16RX (Hitachi Koki, Tokyo, Japan) to collect the lower organic phase. To re-extract the lipids from the water phase, 1 ml chloroform was added. The combined organic phase was evaporated under a nitrogen stream and the extracted lipids were dissolved in methanol.

Sampleprep ID:

SP002441

Sampleprep Summary:

Cells were detached by incubation with trypsin and suspended in PBS. Protein concentration was determined by the bicinchonic acid method (Thermo Fisher Scientific, Rockford, IL). Total lipids were extracted from 50 μg of cellular protein by the Bligh and Dyer method. Cells were dissolved in methanol/chloroform/water at 2:1:0.8 v/v/v and then 50 pmol of 1,2-adidodecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Alabaster, AL) and 1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids) were added as internal standards. After incubation for 5 min at room temperature, 1 ml each of water and chloroform were added and the samples were then centrifuged at 2,000 rpm for 5 min in Himac CF-16RX (Hitachi Koki, Tokyo, Japan) to collect the lower organic phase. To re-extract the lipids from the water phase, 1 ml chloroform was added. The combined organic phase was evaporated under a nitrogen stream and the extracted lipids were dissolved in methanol.

Combined analysis:

Analysis ID	AN003830	AN003831
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity
Column	Waters Aquity BEH C18 (150 x 1.0mm,1.7um)	Waters Aquity BEH C18 (150 x 1.0mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 4000 QTrap	ABI Sciex 4000 QTrap
Ion Mode	POSITIVE	POSITIVE
Units	The ratio to total amount of each phospholipid species	The ratio to total amount of each phospholipid species

Chromatography:

Chromatography ID:	CH002835
Chromatography Summary:	PE and pPE detection method
Instrument Name:	Waters Acquity
Column Name:	Waters Aquity BEH C18 (150 x 1.0mm,1.7um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH002836
Chromatography Summary:	PC and aPC detection method
Instrument Name:	Waters Acquity
Column Name:	Waters Aquity BEH C18 (150 x 1.0mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003572
Analysis ID:	AN003830
Instrument Name:	ABI Sciex 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	MS were detected by neutral loss, precoursour ion scan, and MRM. Peaks were detected by quantification wizard on Ananlyst software (version 1.42).
Ion Mode:	POSITIVE

MS ID:	MS003573
Analysis ID:	AN003831
Instrument Name:	ABI Sciex 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	MS were detected by neutral loss, precoursour ion scan, and MRM. Peaks were detected by quantification wizard on Ananlyst software (version 1.42).
Ion Mode:	POSITIVE