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MB Sample ID: SA096675
| Local Sample ID: | 12_1974_A_M_128_50 |
| 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 |
Select appropriate tab below to view additional metadata details:
Subject:
| 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 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 12_1974_A_M_128_50 | SA096675 | FL013637 | M | Sex |
| 12_1974_A_M_128_50 | SA096675 | FL013637 | 128 | Genotype |
| 12_1974_A_M_128_50 | SA096675 | FL013637 | 50 | Treatment Dosage |
Collection:
| Collection ID: | CO001402 |
| Collection Summary: | For the single injection exposure, mice were sacrificed either 0, 1, 4, 12 or 24 hours after the injection to examine acute changes to Mn. For 1-week and 20-week Mn exposures, mice were sacrificed 24 hours after the final injection. For all studies, mice were sacrificed by cervical dislocation without anesthesia. Mice were decapitated with sharp scissors and the brain removed. The striatum was dissected out under a dissecting microscope, flash-frozen in liquid nitrogen and stored at -80C until later analysis. |
| Sample Type: | Brain |
Treatment:
| Treatment ID: | TR001422 |
| Treatment Summary: | This study utilized three Mn exposure paradigms: 1) a single subcutaneous exposure of either a 1% solution of MnCl2•4(H2O) in filtered MilliQ water at 50 mg/kg body weight or vehicle (filtered water), 2) a 1-week, and 3) 20-week chronic exposure. The 1-week exposure is a previously published week-long exposure protocol (intermittent injections given on days 0, 3 and 6, with sacrifice on day 7) known to elevate brain Mn levels1. The 20-week exposure is a twice weekly injection (Mondays and Thursdays) based on the estimated half-life of 3.5 days for Mn administered via this route and pilot work suggesting the absence of observable toxicity with this exposure paradigm (including no weight loss or physical signs of distress). |
| Treatment Compound: | Mn |
| Treatment Dose: | 0, 5, 15, 50 mg/kg body weight |
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. |
Combined analysis:
| Analysis ID | AN002222 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Vanquish UHPLC binary system |
| Column | SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH001630 |
| Instrument Name: | Vanquish UHPLC binary system |
| Column Name: | SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) |
| Column Temperature: | 40 |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 90% water/10% acetonitrile; 5 mM ammonium formate |
| Solvent B: | 90% acetonitrile/10% water; 5 mM ammonium formate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002068 |
| Analysis ID: | AN002222 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS and UPLC-MS) raw data were imported, processed, normalized and reviewed using Progenesis QI v.2.1 (Non-linear Dynamics, Newcastle, UK). |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Codreags00_20200327_075634_PR_MS_Metabolomics_Protocol.pdf |
