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MB Sample ID: SA259255
Local Sample ID: | 7_A4_C13_Glu_2 |
Subject ID: | SU002689 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002689 |
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 |
---|---|---|---|---|
7_A4_C13_Glu_2 | SA259255 | FL033528 | SMACA4 restoration + C13 Glucose 30min | Treatment |
Collection:
Collection ID: | CO002682 |
Collection Summary: | H1703 with or without SMARCA4/A2 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: | TR002701 |
Treatment Summary: | H1703 with or without SMARCA4/A2 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: | SP002695 |
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 | AN004259 |
---|---|
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: | CH003165 |
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: | MS004006 |
Analysis ID: | AN004259 |
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 |