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MB Sample ID: SA093081
Local Sample ID: | MMV687706_3 |
Subject ID: | SU001346 |
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: | SU001346 |
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 |
---|---|---|---|---|
MMV687706_3 | SA093081 | FL013323 | MMV687706 | Drug treatment |
Collection:
Collection ID: | CO001340 |
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: | TR001361 |
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: | SP001354 |
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 | AN002115 |
---|---|
Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Thermo Dionex Ultimate 3000 RS |
Column | SeQuant ZIC-pHILIC (150 x 4.6mm,5um) |
MS Type | ESI |
MS instrument type | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap |
Ion Mode | UNSPECIFIED |
Units | Peak height |
Chromatography:
Chromatography ID: | CH001548 |
Chromatography Summary: | Metabolite extracts were analysed using hydrophilic interaction (HILIC) liquid chromatography (LC) and high resolution mass spectrometry on an Orbitrap system. |
Chromatography Comments: | Dionex RSLC3000 UHPLC (Thermo). Column additional info: Merck Sequant ZIC-pHILIC 5 µm polymer metal-free(150 x 4.6 mm) |
Instrument Name: | Thermo Dionex Ultimate 3000 RS |
Column Name: | SeQuant ZIC-pHILIC (150 x 4.6mm,5um) |
Column Temperature: | 25°C |
Flow Gradient: | linear gradient -time, %B as follows: 0min- 80%, 15min- 50%, 18min- 5%, 21min- 5%, 24min- 80%, 32min- 80%. |
Flow Rate: | 300 μL/min |
Internal Standard: | internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM) |
Sample Injection: | 10 μL |
Solvent A: | 100% water; 20 mM ammonium carbonate |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS001970 |
Analysis ID: | AN002115 |
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 |
Capillary Voltage: | +50 V |
Spray Voltage: | 4kV |
Resolution Setting: | 35000 |
Scan Range Moverz: | 85- 1275 m/z |