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MB Sample ID: SA099883
Local Sample ID: | WT1 |
Subject ID: | SU001447 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
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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 |
---|---|---|---|---|
WT1 | SA099883 | 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 |