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MB Sample ID: SA096629
| Local Sample ID: | 79_1828_C_F_WT_15 |
| Subject ID: | SU001407 |
| Subject Type: | Other |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | YAC128 and wildtype (WT) |
| Age Or Age Range: | 12 week and 32 week |
| Gender: | Male and female |
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Sample Preparation:
| Sampleprep ID: | SP001415 |
| Sampleprep Summary: | Previously frozen striatal tissue was lysed in 400µL ice-cold lysing buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0) (LC-MS grade). Individual samples were sonicated using a probe tip sonicator, 10 pulses, at 30% power and cooled down on ice between samples. A BCA protein assay was used to determine the protein concentration for each individual sample, and adjusted to a total amount of protein of 200µg in 200 µL of lysis buffer. Isotopically labeled standards, Creatinine-D3 and Lysine-D4, were added to each sample to assess sample processing steps (metabolite extraction and reconstitution). Following lysis and standard addition, protein precipitation was performed by adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate proteins. The supernatants containing metabolites were dried via speed-vacuum. Dried metabolite extracts were stored frozen at -80C until ready to use. Prior to mass spectrometry analysis, extracts were reconstituted in 100 μl of acetonitrile/ water (80:20, v/v) and centrifuged for 5 min at 15K rpm to remove insoluble material. Quality control samples were prepared by pooling equal volumes of each sample. Isotopically labeled standards, Valine-D8 and Inosine-4N15, were added to each sample to determine MS instrument reproducibility. |
| Sampleprep Protocol Filename: | Codreags00_20200327_075634_PR_MS_Metabolomics_Protocol.pdf |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Following lysis and standard addition, protein precipitation was performed by adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate proteins. The supernatants containing metabolites were dried via speed-vacuum. |
| Extract Storage: | -80℃ |
| Sample Resuspension: | 100 μl of acetonitrile/ water (80:20, v/v) and centrifuged for 5 min at 15K rpm to remove insoluble material |
| Sample Spiking: | Valine-D8 and Inosine-4N15, were added to each sample to determine MS instrument reproducibility. |
