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MB Sample ID: SA226974
| Local Sample ID: | MaC1_E1_1 |
| Subject ID: | SU002395 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002395 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| MaC1_E1_1 | SA226974 | FL027552 | C1 | treatment |
| MaC1_E1_1 | SA226974 | FL027552 | 1 | Experiment |
Collection:
| Collection ID: | CO002388 |
| Collection Summary: | For metabolomics experiments, two 150 mL flasks at 6% haematocrit containing tightly synchronised parasites 28-34 hrs post-invasion (5-6 hrs rupture window), were harvested via magnet purification (Miltenyi Biotech). Infected RBC density was quantitated by flow cytometry (Tham et al., 2010) and 2 mL of 3x 107 parasites were added into the wells of 24 well microtiter plates. Parasites were incubated for 1 hrs at 37o C to stabilise the culture. Following this initial incubation, 5x IC50 of the azithromycin analogue C1 and control drugs chloroquine, dihydroartemisinin (DHA) and azithromycin were added and incubated for 2 hrs. Supernatant was removed and parasites washed twice with 800 L ice-cold 1 x PBS, with cells pelleted via centrifugation at 400 x g for 5 mins at 0o C. Cell pellets were resuspended in 200 L of ice-cold extraction buffer (CHCl3/MeOH/water (1:3:1 v/v)) containing 1 µM internal standards, CHAPS and PIPES, and then incubated on ice for 1 hrs with shaking at 200 rpm. Cell debris was pelleted with centrifugation at 14800 x g for 10 mins at 0 oC. The resulting supernatant (180 µL) was transferred to Eppendorf tubes and the remaining ~20 µL were combined to make a pooled QC sample. Extraction blank samples (without cells) were prepared alongside and samples were stored at -80 ºC until analysis. |
| Sample Type: | Blood (whole) |
Treatment:
| Treatment ID: | TR002407 |
| Treatment Summary: | For metabolomics experiments, two 150 mL flasks at 6% haematocrit containing tightly synchronised parasites 28-34 hrs post-invasion (5-6 hrs rupture window), were harvested via magnet purification (Miltenyi Biotech). Infected RBC density was quantitated by flow cytometry (Tham et al., 2010) and 2 mL of 3x 107 parasites were added into the wells of 24 well microtiter plates. Parasites were incubated for 1 hrs at 37o C to stabilise the culture. Following this initial incubation, 5x IC50 of the azithromycin analogue C1 and control drugs chloroquine, dihydroartemisinin (DHA) and azithromycin were added and incubated for 2 hrs. |
Sample Preparation:
| Sampleprep ID: | SP002401 |
| Sampleprep Summary: | Supernatant was removed and parasites washed twice with 800 L ice-cold 1 x PBS, with cells pelleted via centrifugation at 400 x g for 5 mins at 0o C. Cell pellets were resuspended in 200 L of ice-cold extraction buffer (CHCl3/MeOH/water (1:3:1 v/v)) containing 1 µM internal standards, CHAPS and PIPES, and then incubated on ice for 1 hrs with shaking at 200 rpm. Cell debris was pelleted with centrifugation at 14800 x g for 10 mins at 0 oC. The resulting supernatant (180 µL) was transferred to Eppendorf tubes and the remaining ~20 µL were combined to make a pooled QC sample. Extraction blank samples (without cells) were prepared alongside and samples were stored at -80 ºC until analysis. |
Combined analysis:
| Analysis ID | AN003771 | AN003772 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 4.6mm,5um) | SeQuant ZIC-pHILIC (150 x 4.6mm,5um) |
| 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 | relative intensity | relative intensity |
Chromatography:
| Chromatography ID: | CH002790 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 4.6mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 80% B decreasing to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min. |
| Flow Rate: | 0.3 ml/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003514 |
| Analysis ID: | AN003771 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Liquid chromatography-mass spectrometry (LC-MS) data was acquired on a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled with high-performance liquid chromatography system (HPLC, Dionex Ultimate® 3000 RS, Thermo Scientific) as per previously described 49. Briefly, chromatographic separation was performed on a ZIC-pHILIC column equipped with a guard (5 µm, 4.6 × 150 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate (Sigma Aldrich), (B) acetonitrile (Burdick and Jackson) and needle wash solution was 50% isopropanol. The column flow rate was maintained at 0.3 ml/min with temperature at 25 ºC and the gradient program was as follows: 80% B decreasing to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min. Total run time was 32 min with an injection volume of 10 µL. Mass spectrometer was operated in full scan mode with positive and negative polarity switching at 35k resolution at 200 m/z, with detection range of 85 to 1275 m/z, AGC target was 1e6 ions with a maximum injection time of 50 ms. Electro-spray ionization source (HESI) was set to 4.0 kV voltage for positive and negative mode, sheath gas was set to 50, aux gas to 20 and sweep gas to 2 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. The samples were analyzed as a single batch to avoid batch-to-batch variation and randomized to account for LCMS system drift over time. Repeated analysis of pooled quality control samples was performed throughout the batch to confirm signal reproducibility |
| Ion Mode: | POSITIVE |
| MS ID: | MS003515 |
| Analysis ID: | AN003772 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Liquid chromatography-mass spectrometry (LC-MS) data was acquired on a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled with high-performance liquid chromatography system (HPLC, Dionex Ultimate® 3000 RS, Thermo Scientific) as per previously described 49. Briefly, chromatographic separation was performed on a ZIC-pHILIC column equipped with a guard (5 µm, 4.6 × 150 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate (Sigma Aldrich), (B) acetonitrile (Burdick and Jackson) and needle wash solution was 50% isopropanol. The column flow rate was maintained at 0.3 ml/min with temperature at 25 ºC and the gradient program was as follows: 80% B decreasing to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min. Total run time was 32 min with an injection volume of 10 µL. Mass spectrometer was operated in full scan mode with positive and negative polarity switching at 35k resolution at 200 m/z, with detection range of 85 to 1275 m/z, AGC target was 1e6 ions with a maximum injection time of 50 ms. Electro-spray ionization source (HESI) was set to 4.0 kV voltage for positive and negative mode, sheath gas was set to 50, aux gas to 20 and sweep gas to 2 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. The samples were analyzed as a single batch to avoid batch-to-batch variation and randomized to account for LCMS system drift over time. Repeated analysis of pooled quality control samples was performed throughout the batch to confirm signal reproducibility |
| Ion Mode: | NEGATIVE |
