Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA259212

Local Sample ID:	19_A4_C13_Glutamine_4
Subject ID:	SU002688
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU002688
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
19_A4_C13_Glutamine_4	SA259212	FL033511	SMACA4 repstration + C13 Glutamine 60min	Treatment

Collection:

Collection ID:	CO002681
Collection Summary:	BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).
Sample Type:	Ovarian cancer cells

Treatment:

Treatment ID:	TR002700
Treatment Summary:	BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).

Treatment ID:

TR002700

Treatment Summary:

BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).

Sample Preparation:

Sampleprep ID:	SP002694
Sampleprep Summary:	Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade). After 2 rounds 10 min sets of sonication (30 seconds on/30 seconds off at high intensity) on slurry ice using a Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with internal control (800 ng myristic acid-D27) and dried in a cold vacuum centrifuge (Labconco) overnight. The dried pellets were reconstituted with 30 μL of 10 mg/mL methoxyamine-HCl in pyridine, and incubated for 30 min at room temperature. Samples were then derivatized with MTBSTFA for 30 min at 70 °C. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument.

Sampleprep ID:

SP002694

Sampleprep Summary:

Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade). After 2 rounds 10 min sets of sonication (30 seconds on/30 seconds off at high intensity) on slurry ice using a Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with internal control (800 ng myristic acid-D27) and dried in a cold vacuum centrifuge (Labconco) overnight. The dried pellets were reconstituted with 30 μL of 10 mg/mL methoxyamine-HCl in pyridine, and incubated for 30 min at room temperature. Samples were then derivatized with MTBSTFA for 30 min at 70 °C. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument.

Combined analysis:

Analysis ID	AN004258
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890A
Column	Agilent DB5-MS (30m x 0.25mm, 0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5975C
Ion Mode	POSITIVE
Units	intensity

Chromatography:

Chromatography ID:	CH003164
Instrument Name:	Agilent 7890A
Column Name:	Agilent DB5-MS (30m x 0.25mm, 0.25um)
Column Temperature:	60-320
Flow Gradient:	None
Flow Rate:	0.69 ml/min
Solvent A:	None
Solvent B:	None
Chromatography Type:	GC

MS:

MS ID:	MS004005
Analysis ID:	AN004258
Instrument Name:	Agilent 5975C
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument. Metabolites were resolved by separation on DB-5MS+DG (30 m x 250 µm x 0.25 µm) capillary column (Agilent J&W, Santa Clara, CA, USA). Helium was used as the carrier gas with a flow rate such that myristic-d27 acid eluted at approximately 18 min. The quadrupole was set at 150 ˚C, the source was at 230 °C and the GC/MS interface was at 320 ˚C. The oven program started at 60 ˚C held for 1 min, then increased at a rate of 10 ˚C/min until 320 ˚C. Bake-out was at 320 ˚C for 9 min. Metabolites were ionized by electron impact at 70 eV. All samples were injected three times: twice using scan (50-1000 m/z) mode (1x and 25x dilution for steady-state samples or 1x and 24x dilution for tracer samples) and once using selected ion monitoring (SIM) mode. All of the metabolites described in this study were validated against authentic standards confirming mass spectra and retention times. Integration of ion intensities was accomplished using Mass Hunter Quant (Agilent). Generally, M-57+. Ions (and isotopes) were analyzed. Mass isotopomer distribution analysis was carried out using in-house software using an in-house algorithm adapted from Nanchen et al as previously described.
Ion Mode:	POSITIVE