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MB Sample ID: SA258369
| Local Sample ID: | PTEN WT Rep3 |
| Subject ID: | SU002674 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | embryonic fibroblasts |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002674 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | embryonic fibroblasts |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| PTEN WT Rep3 | SA258369 | FL033160 | PTEN Wild-Type | Genotype |
Collection:
| Collection ID: | CO002667 |
| Collection Summary: | PTEN WT and KO MEFs were seeded at 80% in 10cm plates 24 hours prior to metabolite extraction/sample prep |
| Sample Type: | Fibroblasts |
Treatment:
| Treatment ID: | TR002686 |
| Treatment Summary: | media void of cystine was supplemented with 3,3’- 13C2-cystine to 63 mg/L. On day 0, Pten WT and Pten KO MEFs were seeded at 80% confluency in standard media in 10cm plates. After 24 hours, the standard media was replaced with the heavy isotope labeled media for 12 hours before metabolites were extracted as described in the Sampleprep section. N = 5. |
Sample Preparation:
| Sampleprep ID: | SP002680 |
| Sampleprep Summary: | Media was fully removed from the plates. Metabolites were extracted using ice cold 80% methanol and dried using a speed vac. The dried metabolites were sent to the Mass Spec Core ran by John Asara at the Beth Israel Deaconess Medical Center at Harvard University for targeted LC-MS/MS. For detailed sample prep information see the attached protocol. |
Combined analysis:
| Analysis ID | AN004238 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Waters Acquity |
| Column | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| MS Type | APCI |
| MS instrument type | QTRAP |
| MS instrument name | ABI Sciex 5500 QTrap |
| Ion Mode | POSITIVE |
| Units | peak area intensity |
Chromatography:
| Chromatography ID: | CH003145 |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| Column Temperature: | 45 |
| Flow Gradient: | 85% buffer B to 30% buffer B from 0 - 3 minutes, then 30% buffer B to 3% buffer B from minute 3 to 12, then 2% buffer B held from minute 12 to 15, followed by 2% buffer B to 85% buffer B from minute 15 to 16, then 85% buffer B held from minute 16-23 in order to re-equilibrate the column. |
| Flow Rate: | 400uL/minute |
| Solvent A: | 95% water/5% acetonitrile; 20 mM ammonium hydroxide; 20 mM ammonium acetate |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003985 |
| Analysis ID: | AN004238 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | QTRAP |
| MS Type: | APCI |
| MS Comments: | Selective reaction monitoring, SRM, of endogenous water-soluble metabolites was performed for steady state analysis. The positive ion mode ESI voltage was +4900. Dwell time was set to 3ms per SRM transition and 1.55 seconds was the total cycle time. Retention time 15-20 seconds. MultiQuant v2.1 software was used to in integrate peak areas for each metabolite SRM transition |
| Ion Mode: | POSITIVE |
