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MB Sample ID: SA176896

Local Sample ID:	Ding_B-2_008
Subject ID:	SU001987
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	ATF3(+/+) vs ATF3(-/-)
Cell Strain Details:	HCT116 cells engineered to knockout ATF3 expression via rAAV-mediated homologous recombination
Species Group:	Mammals

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Subject:

Subject ID:	SU001987
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	ATF3(+/+) vs ATF3(-/-)
Cell Strain Details:	HCT116 cells engineered to knockout ATF3 expression via rAAV-mediated homologous recombination
Species Group:	Mammals

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Ding_B-2_008	SA176896	FL021934	WT-serine(-)	Genotype_Media supplement

Collection:

Collection ID:	CO001980
Collection Summary:	Cells were counted, washed with cold PBS and then flash-frozen in liquid nitrogen
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR001999
Treatment Summary:	HCT116 cells (~50% confluence) were washed with PBS, and cultured in HEPES buffered Krebs-Ringer solution supplemented with 25 mM [U-13C]-D-glucose, 10% dialysed FBS, 2×MEM amino acids, 2× Vitamin Solution and 4 mM L-glutamine, with or without 0.4 mM serine and glycine, for 24 h. Cells were washed with PBS and metabolites extracted using 1 mL of acetonitrile:isopropanol:water (3:3:2, v/v/v) mixture.

Treatment ID:

TR001999

Treatment Summary:

HCT116 cells (~50% confluence) were washed with PBS, and cultured in HEPES buffered Krebs-Ringer solution supplemented with 25 mM [U-13C]-D-glucose, 10% dialysed FBS, 2×MEM amino acids, 2× Vitamin Solution and 4 mM L-glutamine, with or without 0.4 mM serine and glycine, for 24 h. Cells were washed with PBS and metabolites extracted using 1 mL of acetonitrile:isopropanol:water (3:3:2, v/v/v) mixture.

Sample Preparation:

Sampleprep ID:	SP001993
Sampleprep Summary:	The extraction solvent was degassed and pre-chilled at −20°C. Samples were homogenized using Geno/Grinder 2010 (SPEX SamplePrep) at 1500 rpm for 30s, then shaken at 4°C for 5 min and centrifuged for 2 minutes at 14,000 rcf. 450 μL supernatant was transferred to a new tube and dried down using Centrivap cold trap concentrator (Labconco). 10 μL of methoxyamine hydrochloride in pyridine (40 mg/mL) was added to dried sample and shaken at 30°C for 1.5 hours for methoximation. 90 μL of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA, Sigma-Aldrich) was used for tertbutylsilylation. C8–C30 fatty acid methyl esters (FAMEs) were added to MTBSTFA as internal standards for retention time correction. Samples were shaken at 37°C for 30 min for TMS or shaken at 80°C for 30 min for TBS and then ready for injection.

Sampleprep ID:

SP001993

Sampleprep Summary:

The extraction solvent was degassed and pre-chilled at −20°C. Samples were homogenized using Geno/Grinder 2010 (SPEX SamplePrep) at 1500 rpm for 30s, then shaken at 4°C for 5 min and centrifuged for 2 minutes at 14,000 rcf. 450 μL supernatant was transferred to a new tube and dried down using Centrivap cold trap concentrator (Labconco). 10 μL of methoxyamine hydrochloride in pyridine (40 mg/mL) was added to dried sample and shaken at 30°C for 1.5 hours for methoximation. 90 μL of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA, Sigma-Aldrich) was used for tertbutylsilylation. C8–C30 fatty acid methyl esters (FAMEs) were added to MTBSTFA as internal standards for retention time correction. Samples were shaken at 37°C for 30 min for TMS or shaken at 80°C for 30 min for TBS and then ready for injection.

Combined analysis:

Analysis ID	AN003106
Chromatography ID	CH002293
MS ID	MS002888
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890A
Column	Restek Rtx-5Sil (30m x 0.25mm,0.25um)
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Agilent 7890A
Ion Mode	UNSPECIFIED
Units	enrichment percentage

Chromatography:

Chromatography ID:	CH002293
Chromatography Summary:	Gas chromatography-Quadrupole-time of flight mass spectrometry (GC-Q-TOF MS) was used for Stable Isotope enrichment. Rtx-5Sil MS column (30m length, 0.25 mm i.d, 0.25 μM 95% dimethyl 5% diphenyl polysiloxane film) with an additional 10 m guard column was installed on Agilent 7890 GC (Agilent Technologies). 99.9999% pure Helium gas was used as a mobile phase with a flow rate of 1mL/min. GC temperature was held at 50°C for 1 min, ramped at 20°C/min to 330°C and then held for 5 min. Electron ionization at -70eV was employed on a Agilent 7250 Quadrupole time of flight mass spectrometer (Agilent Technologies) with ion source temperature at 250°C and detector voltage at 1850V. Mass spectra were acquired at an acquisition rate of 5 spectra/s, with a scan range of 85-900 Da.
Instrument Name:	Agilent 7890A
Column Name:	Restek Rtx-5Sil (30m x 0.25mm,0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS002888
Analysis ID:	AN003106
Instrument Name:	Agilent 7890A
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	Agilent MassHunter Quantitative Analysis software B.10.00 (Agilent Technologies) was used for post data processing.
Ion Mode:	UNSPECIFIED