Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA098898

Local Sample ID:	VV_17_HEpG2_SDC2
Subject ID:	SU001433
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Biosource Or Supplier:	HPACC
Cell Passage Number:	10-20

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Subject:

Subject ID:	SU001433
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Biosource Or Supplier:	HPACC
Cell Passage Number:	10-20

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
VV_17_HEpG2_SDC2	SA098898	FL014026	SDC_i	Variable

Collection:

Collection ID:	CO001428
Collection Summary:	Single-phase lipid extraction was performed according to some reported protocols for metabolomics extraction (MacKay et al., 2015). Briefly, media was quickly removed from the plates and each well was washed twice with ice-cold PBS. An extracting solution containing either isopropanol (IPA) or a mixture 1:1 of methanol-butanol (BuMe), kept at 4°C, was used to simultaneously quench all metabolic reactions and to extract intra-cellular lipids. Plates were incubated on dry ice for 20 min and extracted lipids were transferred to 1.5 ml Eppendorf tubes (Stevenage, UK) followed by centrifugation (4°C) at 14000 rpm for 10min to remove the denatured proteins. Supernatants were collected and stored at -80°C prior to LC-MS analysis. Protein pellets attached to the plate were left to dry overnight for the subsequent protein normalisation by the modified-Lowry procedure (MacKay et al., 2015).
Collection Protocol Filename:	garodriguezblanco10_20200326_110242_PR_CO_Methods.docx
Sample Type:	HepG2 cells

Treatment:

Treatment ID:	TR001448
Treatment Summary:	HepG2 cells were treated with a desaturase inhibitor for 24h.

Sample Preparation:

Sampleprep ID:	SP001441
Sampleprep Summary:	Single-phase lipid extraction was performed according to some reported protocols for metabolomics extraction (MacKay et al., 2015). Briefly, media was quickly removed from the plates and each well was washed twice with ice-cold PBS. An extracting solution containing either isopropanol (IPA) or a mixture 1:1 of methanol-butanol (BuMe), kept at 4°C, was used to simultaneously quench all metabolic reactions and to extract intra-cellular lipids. Plates were incubated on dry ice for 20 min and extracted lipids were transferred to 1.5 ml Eppendorf tubes (Stevenage, UK) followed by centrifugation (4°C) at 14000 rpm for 10min to remove the denatured proteins. Supernatants were collected and stored at -80°C prior to LC-MS analysis. Protein pellets attached to the plate were left to dry overnight for the subsequent protein normalisation.

Sampleprep ID:

SP001441

Sampleprep Summary:

Single-phase lipid extraction was performed according to some reported protocols for metabolomics extraction (MacKay et al., 2015). Briefly, media was quickly removed from the plates and each well was washed twice with ice-cold PBS. An extracting solution containing either isopropanol (IPA) or a mixture 1:1 of methanol-butanol (BuMe), kept at 4°C, was used to simultaneously quench all metabolic reactions and to extract intra-cellular lipids. Plates were incubated on dry ice for 20 min and extracted lipids were transferred to 1.5 ml Eppendorf tubes (Stevenage, UK) followed by centrifugation (4°C) at 14000 rpm for 10min to remove the denatured proteins. Supernatants were collected and stored at -80°C prior to LC-MS analysis. Protein pellets attached to the plate were left to dry overnight for the subsequent protein normalisation.

Combined analysis:

Analysis ID	AN002263
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Ultimate 3000
Column	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	Normalised peak area

Chromatography:

Chromatography ID:	CH001662
Chromatography Summary:	The mobile phases consisted of 60:40 ACN: H2O with 10mM ammonium formate, 0.1% formic acid and 5µM of phosphoric acid (A) and 90:10 IPA:ACN with 10 mM ammonium formate, 0.1% formic acid and 5µM phosphoric acid (B). The gradient was as follows: 0-2 min 30% (B); 2-8 min 50% (B); 8-15 min 99% (B), 15-16 min 99% (B), 16-17 min 30% (B). Sample temperature was maintained at 6°C in the autosampler and 5 µL of sample were injected into the LC-MS instrument.
Instrument Name:	Ultimate 3000
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Flow Gradient:	0-2 min 30% (B); 2-8 min 50% (B); 8-15 min 99% (B), 15-16 min 99% (B), 16-17 min 30% (B).
Solvent A:	60% acetonitrile/40% water; 0.1% formic acid; 10mM ammonium formate; 5µM phosphoric acid
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate;5µM phosphoric acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002107
Analysis ID:	AN002263
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Thermo Q-Exactive Orbitrap MS instrument was operated in both positive and negative polarities, using the following parameters: mass range 240-1200 m/z (positive) and 240-1600 (negative), spray voltage 3.8kV (ESI+) and 3kV (ESI-), sheath gas (nitrogen) flow rate 60 units, auxiliary gas (nitrogen) flow rate 25 units, capillary temperature (320°C), full scan MS1 mass resolving power 70,000. Data dependent fragmentation (dd-MS/MS) parameters for each polarity as follows: TopN: 10, resolution 17,500 units, maximum injection time: 25 ms, automatic gain control target: 5e5 and normalised collision energy of 20 and 25 (arbitrary units) in positive polarity. TopN: 5, resolution 17,500 units, maximum injection time: 80 ms automatic gain control target: 5e5 and normalised collision energy of 20 and 30 (arbitrary units) in negative polarity. The instrument was externally calibrated to <1ppm using ESI positive and negative calibration solutions (Thermo Fisher Scientific).
Ion Mode:	POSITIVE