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MB Sample ID: SA185815
| Local Sample ID: | 20190426-183P_2.4_1 |
| Subject ID: | SU002066 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | 3D7 |
| Cell Counts: | 1*10^8 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002066 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | 3D7 |
| Cell Counts: | 1*10^8 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 20190426-183P_2.4_1 | SA185815 | FL022982 | 20190426-183P_2.4 | Factor |
Collection:
| Collection ID: | CO002059 |
| Collection Summary: | Parasites were magnetically purified at the early trophozoite stage from cultures of 5-10% parasitemia. Samples were generated with 5 mL of media containing 1*10^8 cells (either parasites or human red blood cells) for the treatment condition. Excess media was removed and the cells were centrifuged and washed once with PBS. Metabolism was quenched with 90% methanol containing 0.25 micromolar of universally labeled C-13, N-15 aspartate. The samples were centrifuged and the metabolites in the methanol were collected and transferred to a new tube to be dried with nitrogen. |
| Collection Protocol Filename: | Metabolite_Extraction_for_LCMS_2017.pdf |
| Sample Type: | Cultured cells |
| Collection Location: | Millenium Science Complex, University Park, Pennsylvania |
| Storage Conditions: | -80℃ |
| Collection Vials: | 1.5 mL eppendorf |
| Storage Vials: | 1.5 mL eppendorf |
| Collection Tube Temp: | On ice |
| Tissue Cell Quantity Taken: | 1x10^8 cells per sample in 1 mL total volume |
Treatment:
| Treatment ID: | TR002078 |
| Treatment Summary: | In all conditions 1*10^8 cells were cultured in 5 mL of RPMI 1640 media for 2.5 hours. At the start of the incubation period, cells were either not exposed to any drug or exposed to 240 nM, 24 nM, or 2.4 nM MMV693183. Dates reflect date of running an individual sample on the analytical platform. |
| Treatment Compound: | MMV693183 |
| Treatment Route: | Transfer to media by pipette |
| Treatment Dose: | 5 microliters of drug to a final concentration of 240 nM, 24 nM, or 2.4 nM. |
| Treatment Vehicle: | DMSO |
| Cell Growth Container: | 6-well plate |
| Cell Growth Config: | 5 mL in each sample well, 1x10^8 infected red blood cells per well, conditions performed in triplicate |
| Cell Media: | RPMI 1640 containing Albumax, Gentamycin, Hypoxanthine, HEPES, Sodium Bicarbonate |
Sample Preparation:
| Sampleprep ID: | SP002072 |
| Sampleprep Summary: | Once samples were obtained, metabolism was quenched with 90% methanol containing 0.25 uM labeled aspartate, cells were centrifuged, and the supernatant was removed by nitrogen drying. Sample was reconstituted in 3% HPLC-grade methanol and run on the instrument. |
| Processing Method: | Wash, spin, quench, spin, dry, store at -80 C until run on the instrument, resuspend |
| Processing Storage Conditions: | On ice |
| Extraction Method: | 90% methanol extraction |
| Extract Cleanup: | Centrifugation and nitrogen drying |
| Sample Resuspension: | 100 uL 3% methanol with 1 uM chlorpropamide |
| Sample Spiking: | 0.25 uM Labelled Aspartate, 1 uM chlorpropamide |
Combined analysis:
| Analysis ID | AN003236 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Waters XSelect HSS (100 x 2.1mm,2.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | Peak Abundance (normalized, blank subtracted, and corrected for baseline noise) |
Chromatography:
| Chromatography ID: | CH002386 |
| Chromatography Summary: | Ion-pairing method using reverse-phase chromatography setup. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters XSelect HSS (100 x 2.1mm,2.5um) |
| Column Temperature: | 30 |
| Flow Rate: | 0.200 mL/minute |
| Solvent A: | 97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003009 |
| Analysis ID: | AN003236 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data was centroided using MSConvert and converted to .mzXML for utilization in MzMine and El-Maven software. |
| Ion Mode: | NEGATIVE |
