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MB Sample ID: SA099879
| Local Sample ID: | WT5 |
| Subject ID: | SU001447 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001447 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| WT5 | SA099879 | FL014134 | Wild-type | Genotype |
Collection:
| Collection ID: | CO001442 |
| Collection Summary: | CD8+ purified T cells from WT (n = 5) and Sirt2 KO (n = 4) mouse spleens were stimulated with plate-coated anti-CD3 (5 μg/ml, BXCELL) for 72h, followed by extensive washing of the cell pellets with PBS. All processes are carried out on ice. An aliquot (1 mL) of 80% MeOH extraction solvent is added to the sample (with Internal Standards spiked in) for protein precipitation. The 80% MeOH extraction solvent is is precooled to -80C for at least one hour. After addition of the extraction solvent, the samples are then placed in the -80C freezer for 30 minutes. After incubation, the samples are immediately centrifuged at 18,800 × g (Microfuge 22R, Beckman Coulter) at 4 C for 10 minutes. After centrifugation, the supernatant is transferred to new a microcentrifuge tube (Eppendorf brand) for drying by vacuum concentration. The dried metabolites are re-dissolved in 80% MeOH. The precipitated protein pellet is resolubilized for Bradford assays to measure the protein concentration as a quality control for the sample loading. |
| Sample Type: | Spleen |
| Collection Method: | T-cell isolation |
| Collection Location: | Moffitt Cancer Center |
| Collection Frequency: | 1 time |
| Volumeoramount Collected: | 2,000,000 cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001462 |
| Treatment Summary: | CD8+ purified T cells from WT (n = 5) and Sirt2 KO (n = 4) mouse spleens were stimulated with plate-coated anti-CD3 (5 μg/ml, BXCELL) for 72h, followed by extensive washing of the cell pellets with PBS. |
| Treatment: | anti-CD3 stimulation |
| Treatment Compound: | antibody |
| Treatment Route: | Cells placed on coated plate |
| Treatment Doseduration: | 72 hours |
Sample Preparation:
| Sampleprep ID: | SP001455 |
| Sampleprep Summary: | All processes are carried out on ice. An aliquot (1 mL) of 80% MeOH extraction solvent is added to the sample (with Internal Standards spiked in) for protein precipitation. The 80% MeOH extraction solvent is precooled to -80C for at least one hour. After addition of the extraction solvent, the samples are then placed in the -80 oC freezer for 30 minutes. After incubation, the samples are immediately centrifuged at 18,800 × g (Microfuge 22R, Beckman Coulter) at 4 oC for 10 minutes. After centrifugation, the supernatant is transferred to new a microcentrifuge tube (Eppendorf brand) for drying by vacuum concentration. The dried metabolites are re-dissolved in 80% MeOH. The precipitated protein pellet is resolubilized for Bradford assays to measure the protein concentration as a quality control for the sample loading. |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Methanol |
| Extract Enrichment: | Vacuum centrifugation |
| Sample Resuspension: | 80% MeOH |
| Sample Derivatization: | None |
| Sample Spiking: | Internal Standards added |
| Subcellular Location: | N/A |
Combined analysis:
| Analysis ID | AN002293 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Vanquish |
| Column | SeQuant ZIC-pHILIC |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE |
| Units | Peak Intensity |
Chromatography:
| Chromatography ID: | CH001684 |
| Chromatography Summary: | UHPLC-MS was performed using a Vanquish LC (Thermo, San Jose, CA) interfaced with a Q Exactive HF mass spectrometer (Thermo, San Jose, CA). Chromatographic separation was performed on a SeQuant ZIC-pHILIC LC column (150 × 4.6 mm, 5 µm particle size, MilliporeSigma, Burlington, MA). In order to maintain the stable column pressure and further filter the LC solvents, a SeQuant ZIC-pHILIC guard column (20 × 4.6 mm, 5 µm particle size, MilliporeSigma, Burlington, MA) is connected before the LC column. The mobile phase A was aqueous 10 mM ammonium carbonate and 0.05% ammonium hydroxide, and the mobile phase B was 100% acetonitrile. The total running time is 20 minutes. The column temperature was set to 30°C, and the injection volume is 2 µL. |
| Instrument Name: | Vanquish |
| Column Name: | SeQuant ZIC-pHILIC |
| Column Temperature: | 30 |
| Flow Gradient: | 80% B to 20%B over 13 minutes |
| Flow Rate: | 0.25 ml/min |
| Injection Temperature: | 5 |
| Internal Standard: | D-Glucose (2,3,4,5,6-13C5), D-Glucose-6-phosphate (U-13C6), D-Fructose-1, 6-bisphosphate (U-13C6), L-Serine (13C3), Glycine (1,2-13C2), L-Cysteine (3,3-D2), Phosphoenol Pyruvate (2,3-13C2), Lactate (3,3,3-D3), Pyruvate (D3), Acetyl-1,2-13C2 CoA, Citric Acid (2,2,4,4-D4), Alpha-Ketoglutaric Acid (1,2,3,4-13C4), Succinic Acid (D4), Fumaric Acid (D4), DL-Malic Acid (2,3,3-D3), D-Fructose-6-phosphate (U-13C6) are all from Cambridge Isotope Labs. |
| Solvent A: | 100% water; 10 mM ammonium carbonate; 0.05% ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 20 |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002137 |
| Analysis ID: | AN002293 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Full MS is performed in positive and negative mode separately, and the mass scan range is 60 to 900 m/z. In addition to MS data acquisition 1, parallel reaction monitoring (PRM) data are acquired for the important intermediates involved in Glycolysis and the TCA cycle. |
| Ion Mode: | NEGATIVE |
