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MB Sample ID: SA005169
| Local Sample ID: | MaleVsFemale_UT_Female_Caucasian_23_BRH600214_A |
| Subject ID: | SU000111 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and Female |
| Human Race: | Hispanic, Aisan, Caucasian |
| Species Group: | Human |
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Sample Preparation:
| Sampleprep ID: | SP000107 |
| Sampleprep Summary: | concomitant protein precipitation with cold methanol, vortexing, centrifugation, supernatent dried in vacuo, stored at -80° c, chemical derivitization |
| Sampleprep Protocol Comments: | Metabolites were extracted with concomitant protein precipitation by addition of 1 mL of cold (-20 C) methanol with vortexing for 30 s, and precipitated proteins were removed by centrifugation at 15,000xg for 10 min at 4 C. The supernatants were transferred to glass autosampler vials and then dried in vacuo prior to chemical derivatization. If the extracts could not be immediately derivatized an d analyzed by GCMS, then they were stored at -80C. Dried metabolite extracts were chemically derivatized using a modified version of the protocol used to create FiehnLib (Kind et al., 2009). Briefly, dried metabolite extracts were dried again to remove any residual water if they had been stored at -80°C. To protect carbonyl groups and reduce the number of tautomeric isomers, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by vortexing for 30 s and incubation at 37°C with generous shaking (1000 rpm) for 90 min. At this point, the sample vials were inverted one time to capture any condensation of solvent at the cap surface, followed by a brief centrifugation at 1000×g for 1 min. To derivatize hydroxyl and amine groups to trimethylsilyated (TMS) forms, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were then added to each vial, followed by vortexing for 10 s and incubation at 37°C with shaking (1000 rpm) for 30 min. Again, the sample vials were inverted one time, followed by centrifugation at 1000×g for 5 min. The samples were allowed to cool to room temperature and were analyzed in the same day. |
| Processing Method: | precipitation, centrifugation |
| Processing Storage Conditions: | dried in vacuo, stored at -80° C |
| Extraction Method: | concomitant protein precipitation by addition of 1 mL of cold (-20 C) methanol with vortexing and centrifugation |
| Extract Enrichment: | dried in vacuo |
| Extract Storage: | -80° C |
| Sample Resuspension: | 20 µL of methoxyamine in pyridine (30 mg/mL) |
| Sample Derivatization: | 20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) |
