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MB Sample ID: SA099432
| Local Sample ID: | P12_t0 |
| Subject ID: | SU001443 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
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Sample Preparation:
| Sampleprep ID: | SP001451 |
| Sampleprep Summary: | For LC-MS, proteins were removed adding 300 µL of cold (-20 °C) methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and stored on ice for 5 min. Supernatant containing the metabolites was separated from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into a LC vial for analysis. For GC-MS, serum samples were first deproteinized using cold acetonitrile (ACN) in a 3:1 proportion (120 μL of ACN were added to 40 μL of serum). Samples were kept on ice for 5 minutes. Afterwards, the metabolites were separated by centrifugation (10 min at 15,400 x g and 4°C). 100 µL of the resulting supernatant were transferred to a GC vial with insert and were evaporated to dryness for 2 hours at 30°C (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, a methoximation was performed by addition of 10 µL of O-methoxyamine hydrochloride 15mg/mL in pyridine, with the purpose of protecting the reactive oxygen groups in the metabolites. The mixture was vigorously vortex-mixed (1 minute each vial), ultrasonicated (3 times, 20 seconds each) and further vortexed another 2 minutes. Then, the samples were left in darkness at room temperature for 16 h for the completion of the methoximation reaction. Finally, the samples were derivatized by the addition of 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS). |
| Processing Method: | Protein precipitation and metabolite extraction |
| Processing Storage Conditions: | On ice |
