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MB Sample ID: SA093187
| Local Sample ID: | MMV689028_2 |
| Subject ID: | SU001347 |
| Subject Type: | Cultured cells |
| Subject Species: | Trypanosoma brucei brucei |
| Taxonomy ID: | 5702 |
| Genotype Strain: | 427 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001347 |
| Subject Type: | Cultured cells |
| Subject Species: | Trypanosoma brucei brucei |
| Taxonomy ID: | 5702 |
| Genotype Strain: | 427 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| MMV689028_2 | SA093187 | FL013352 | MMV689028 | Drug treatment |
Collection:
| Collection ID: | CO001341 |
| Collection Summary: | Trypanosoma brucei brucei (T.b.b) bloodstream forms (strain 427) were maintained in vitro in 5-10 ml cultures at 37 °C and 5% CO2 in Creeks minimal media supplemented with 10% HMI-9. The cultures were passaged every 2-3 days and cells were grown to a maximum density of 2x106cells/ml. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR001362 |
| Treatment Summary: | For drug-induced metabolic perturbation experiments, cells were sub-cultured at 10e6cells/ml in 20 ml volume with drugs added at 0.5 µM concentration and incubated for a further 5 hours until cell density reached ~2x10e6cells/ml. Cells were then used for metabolite quenching and extraction. |
Sample Preparation:
| Sampleprep ID: | SP001355 |
| Sampleprep Summary: | Metabolism was rapidly quenched by rapidly cooling cultures to 4°C in a dry ice and ethanol bath. Cultures were then centrifuged for 10 minutes at 1250g at 4°C. The supernatant was discarded and cells were washed with 1 ml of cold PBS by centrifugation for 1 minute at 2100g at 4°C. The supernatant was removed and cells were extracted with 100 µl extraction solvent containing chloroform: methanol: water (1:3:1 v/v) followed by vortexing at 4 °C for 1 hour. The resulting suspension was centrifuged for 10 minutes at 2100g at 4°C and the supernatant was transferred to glass vials and stored at -80 °C until analysis by liquid chromatography and high-resolution mass spectrometry. |
Combined analysis:
| Analysis ID | AN002116 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS |
| Column | Ascentis Express C8 (10cm x 2.1mm,2.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Peak height |
Chromatography:
| Chromatography ID: | CH001549 |
| Chromatography Summary: | Metabolite extracts were analysed using reversed phase liquid chromatography (LC) and high resolution mass spectrometry on an Orbitrap system. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Ascentis Express C8 (10cm x 2.1mm,2.7um) |
| Column Temperature: | 40°C |
| Flow Gradient: | linear gradient -time, %B as follows: 0min- 0%, 1.5min- 20%, 7min- 28%, 8min- 35%, 24min- 65%, 25min- 100%. |
| Flow Rate: | 200 μL/min |
| Sample Injection: | 10 μL |
| Solvent A: | 40% isopropanol/60% water; 2 mM formic acid; 8 mM ammonium formate |
| Solvent B: | 98% isopropanol/2% water; 2 mM formic acid; 8 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001971 |
| Analysis ID: | AN002116 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The raw LC-MS data was processed using IDEOM. |
| Ion Mode: | UNSPECIFIED |
| Capillary Temperature: | 300°C |
| Spray Voltage: | 3.5kV |
| Resolution Setting: | 70000 |
| Scan Range Moverz: | 140-1300 m/z |
