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MB Sample ID: SA213105
| Local Sample ID: | IgEki (OVA_EW) 5S |
| Subject ID: | SU002322 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
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Sample Preparation:
| Sampleprep ID: | SP002328 |
| Sampleprep Summary: | FAECES. Frozen faeces samples were lyophilized overnight at -80°C. Faecal pellets were doubly extracted (i) first with MeOH:MTBE (4:1, v/v) and (ii) with MeOH:H2O (4:1, v/v). Equal volumes (300 µL) of the supernatants from (i) and (ii) were then mixed and aliquoted for subsequent analysis. SERUM. Serum samples (100 µL) were deproteinized by an addition of 300 µL of cold acetonitrile (1:3 v/v) and homogenized for 15 min in a vortex. After a 10 min ice bath, samples were centrifuged (16000 x g, 4°C, 20 min) and stored at -20°C until the start of sample treatment. The resulting supernatants were filtered through 0.22 µm for LC-MS, 80 µL were transferred into an analytical vial for their analysis. For CE-MS, 80 µL of the supernatants were evaporated to dryness using a Speedvac Concentrator and reconstituted in 80 µL of MilliQ® water containing an internal standard (0.2 mM L-methionine sulfone) and 0.1 M FA. Finally, for GC-MS, 120 µL of the corresponding aliquots were evaporated to dryness using a Speedvac Concentrator, followed by the addition of 10 µL of O-methoxyamine hydrochloride (15 mg/mL) in pyridine for methoximation. After gently vortexing, the vials were incubated in darkness at room temperature for 16 h. Then, 10 µL of BSTFA with 1 % TMCS (v/v) were added and samples were vortexed for 5 min, silylation was carried out for 1 h at 70 °C and finally 120 µL of C18:0 methyl ester (10 mg/L in heptane) were added as an internal standard and samples were mixed again by gentle vortexing. Eight blank samples were prepared for GC-MS by the same procedure of extraction and derivatization. |
| Sampleprep Protocol Filename: | Protocolsamples.docx |
| Processing Method: | Lyophilization, homogenization, protein precipitation and metabolite extraction |
| Processing Storage Conditions: | On ice |
