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MB Sample ID: SA020681
| Local Sample ID: | ciMB1_H05b |
| Subject ID: | SU000435 |
| Subject Type: | Cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | 3D7 |
| Species Group: | Microorganism |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000435 |
| Subject Type: | Cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | 3D7 |
| Species Group: | Microorganism |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| ciMB1_H05b | SA020681 | FL004894 | MB1_H05 | Drug |
Collection:
| Collection ID: | CO000429 |
| Collection Summary: | Asexual P. falciparum (3D7) were cultured with minor modifications to the method of Trager and Jensen using human RBC (Australian Red Cross Blood Service) at 3% hematocrit in modified RPMI medium containing hypoxanthine and 0.5% (w/v) albumax at 37 °C under defined atmosphere (95% N2, 4% CO2, 1% O2). Parasites were synchronized with 5% (w/v) sorbitol twice at an interval of 14 hours and cultured for a further 58 hours to ensure all experiments were performed on mid-trophozoite stage cultures (~30 h post infection) at 7-8% parasitaemia. |
| Sample Type: | Cell |
Treatment:
| Treatment ID: | TR000449 |
| Treatment Summary: | Cultures (200 µL) were incubated with test compounds (1 µM) for 5 hours in 96-well plates. Four replicate incubations of each compound were conducted and analyzed. Untreated controls contained equivalent amounts of DMSO (as vehicle), and additional ‘quench test’ controls were prepared whereby the test compounds were added after the quenching step, to allow detection of test compound-derived LC-MS features that did not arise from biochemical metabolism within the cells. Each plate contained 10 or 20 test compounds in addition to positive controls (artemisinin and/or atovaquone) and negative controls (untreated DMSO and ‘quench test’ described above). Incubations and extractions for the 100 test compounds (10 compounds with known antimalarial activity and 90 of the highest priority compounds from the Malaria Box including all of plate A and the first row of plate B) were performed in three separate batches, with the known antimalarial compounds and first 10 Malaria Box compounds in the first batch, the next 40 Malaria Box compounds in the second batch, and the following 40 Malaria Box compounds in the third batch. |
| Treatment Compound: | Antimalarials and MMV Malaria Box compounds |
| Treatment Dose: | 1 micromolar |
| Treatment Doseduration: | 5 hours |
| Treatment Vehicle: | DMSO |
| Cell Growth Container: | 96-well V-bottomed plate |
| Cell Media: | RPMI-HEPES |
| Cell Envir Cond: | 37 °C under defined atmosphere (95% N2, 4% CO2, 1% O2) |
| Cell Media Lastchanged: | 18 hours |
Sample Preparation:
| Sampleprep ID: | SP000442 |
| Sampleprep Summary: | Culture medium was removed by aspiration, and the metabolism of the settled iRBCs quenched by addition of ice-cold phosphate buffered saline (PBS). Subsequent steps were performed on ice. Cells were pelleted by centrifugation for 5 min at 1000 × g and the PBS supernatant removed prior to the addition of 135 µL methanol (containing internal standard compounds: CHAPS, CAPS and PIPES) and rapid mixing by pipetting three times to extract iRBC metabolites. Samples were left on ice with gentle agitation for 60 minutes, then centrifuged at 3000 × g to remove the insoluble material. Supernatants were transferred to glass HPLC vials and stored (< 4 months) at -80 °C until analysis. |
| Processing Method: | Lysis with organic solvent and mixing at 4°C |
| Processing Storage Conditions: | on ice or 4°C |
| Extraction Method: | Methanol (10:1) |
| Extract Storage: | -80°C |
| Sample Spiking: | internal standards (CHAPS, CAPS, PIPES all at 1 µM) mixed in extraction solvent |
| Cell Type: | Plasmodium falciparum-infected red blood cells (8% parasitaemia and 3% haematocrit |
Combined analysis:
| Analysis ID | AN000655 | AN000656 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | ZIC-pHILIC (Merck Sequant) | ZIC-pHILIC (Merck Sequant) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak height | Peak height |
Chromatography:
| Chromatography ID: | CH000473 |
| Chromatography Summary: | Untargeted HILIC method |
| Methods Filename: | pHILIC_32min_posneg_71114.meth |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | ZIC-pHILIC (Merck Sequant) |
| 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 |
| Injection Temperature: | 4 °C |
| Internal Standard: | internal standards (CHAPS, CAPS, PIPES; 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: | MS000581 |
| Analysis ID: | AN000655 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 300°C |
| Capillary Voltage: | +50V |
| Dry Gas Flow: | 20 |
| Fragmentation Method: | None |
| Ion Spray Voltage: | 3.5kV |
| Desolvation Gas Flow: | 50 |
| Desolvation Temperature: | 150°C |
| Resolution Setting: | 35000 |
| Scan Range Moverz: | 85- 1275 m/z |
| MS ID: | MS000582 |
| Analysis ID: | AN000656 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 300°C |
| Capillary Voltage: | -50V |
| Dry Gas Flow: | 20 |
| Fragmentation Method: | None |
| Ion Spray Voltage: | 3.5kV |
| Desolvation Gas Flow: | 50 |
| Desolvation Temperature: | 150°C |
| Resolution Setting: | 35000 |
| Scan Range Moverz: | 85- 1275 m/z |
