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MB Sample ID: SA084768
| Local Sample ID: | 5h_OZ439_S_troph_c |
| Subject ID: | SU001272 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum;Homo sapiens |
| Taxonomy ID: | 5833;9606 |
| Genotype Strain: | Cam3.IIR539T and Cam3.IIrev |
| Age Or Age Range: | 22-26 h post invasion |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001272 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum;Homo sapiens |
| Taxonomy ID: | 5833;9606 |
| Genotype Strain: | Cam3.IIR539T and Cam3.IIrev |
| Age Or Age Range: | 22-26 h post invasion |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 5h_OZ439_S_troph_c | SA084768 | FL012817 | Cam3.IIrev | parasite_line |
| 5h_OZ439_S_troph_c | SA084768 | FL012817 | OZ439 | treatment |
| 5h_OZ439_S_troph_c | SA084768 | FL012817 | 5 | treatment_duration_(h) |
Collection:
| Collection ID: | CO001266 |
| Collection Summary: | RBCs infected with Cam3.IIR539T (resistant) or Cam3.IIrev (sensitive) P. falciparum parasites (22-26 h post invasion) were adjusted to 4% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 1E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 150 µL of cold methanol. The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR001287 |
| Treatment Summary: | RBCs infected with Cam3.IIR539T (resistant) or Cam3.IIrev (sensitive) P. falciparum parasites (22-26 h post invasion) at 4% parasitaemia and 2% Hct were treated with 100 nM of DHA, OZ277 or OZ439 (or an equivalent volume of DMSO) for 1, 3 and 5 h, respectively. During the drug incubation period parasite cultures were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2. |
| Treatment Compound: | OZ277 (arterolane), OZ439 (artefenomel) and dihydroartemisinin (DHA) |
| Treatment Vehicle: | DMSO |
| Cell Media: | Complete RPMI medium (10.4 g/L) containing HEPES (5.94 g/L), hypoxanthine (50 mg/L), sodium bicarbonate (2.1 g/L) and Albumax II (5 g/L). |
| Cell Media Lastchanged: | Immediately prior to initiation of drug incubation |
Sample Preparation:
| Sampleprep ID: | SP001280 |
| Sampleprep Summary: | RBCs infected with Cam3.IIR539T (resistant) or Cam3.IIrev (sensitive) P. falciparum parasites (22-26 h post invasion) were adjusted to 4% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 1E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 150 µL of cold methanol. The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample. |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | Described in summary |
Combined analysis:
| Analysis ID | AN002006 | AN002007 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | SeQuant ZIC-pHILIC (150 x 2.1mm,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 | Peak intensity | Peak intensity |
Chromatography:
| Chromatography ID: | CH001451 |
| Chromatography Summary: | The 32 min gradient HPLC run was from 80% B to 50% B over 15 min, then to 5% B at 18 min, followed by a wash with 5% B for 3 min and re-equilibrated with 80% B at a flow rate of 0.3 mL/min. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS001859 |
| Analysis ID: | AN002006 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. LC MS peak heights representing metabolite abundances were normalised by median peak height. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times. |
| Ion Mode: | POSITIVE |
| MS ID: | MS001860 |
| Analysis ID: | AN002007 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. LC MS peak heights representing metabolite abundances were normalised by median peak height. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times. |
| Ion Mode: | NEGATIVE |
