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MB Sample ID: SA189644
| Local Sample ID: | 20190313-WT_glutamine-3 |
| Subject ID: | SU002106 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | NF54 attB |
| Gender: | Not applicable |
| Cell Counts: | 1x10^8 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002106 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | NF54 attB |
| Gender: | Not applicable |
| Cell Counts: | 1x10^8 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 20190313-WT_glutamine-3 | SA189644 | FL023419 | 20190313-WT_glutamine | Treatment |
Collection:
| Collection ID: | CO002099 |
| Collection Summary: | Plasmodium falciparum NF54 attB parasites containing different genetic backgrounds were cultured in RPMI 1640 medium and magnetically enriched to increase the infected to uninfected RBC ratio. Following hemocytometer counts, 1x10^8 parasites were measured per condition into 5 mL of total media for 2.5 hours in the presence of the universally labelled carbon-13 metabolite (glucose, glutamine, or acetate). Medium was aspirated until 1 mL remained on each sample, the sample was transferred to a micro-centrifuge tube, spun, and the medium was aspirated. |
| 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: | TR002118 |
| Treatment Summary: | Plasmodium falciparum NF54 attB parasites were cultured in RPMI 1640 medium and magnetically enriched to increase the infected to uninfected RBC ratio. Following hemocytometer counts, 1x10^8 parasites were measured per condition into 5 mL of total media for 2.5 hours in the presence of the carbon-13 metabolite (glucose, glutamine, or acetate). Wild-type or WT represents control parasites without additional alterations. Blanks are sample tubes that follow the same procedures as samples following the quenching of metabolism. Both Pool and QC samples are combined samples of all samples from the analytical batch on that particular day. Dates in YYYYMMDD format are appended to individual samples to indicate the batch in which they were processed. |
| Treatment Protocol Filename: | Metabolite_Extraction_for_LCMS_2017.pdf |
| Treatment Compound: | Universally labelled glucose, glutamine, or acetate |
| Treatment Route: | Transfer to media by pipette |
| Treatment Dosevolume: | 50 uL (glucose), 25 uL (glutamine), or 10 uL (acetate) |
| Treatment Doseduration: | 2.5 hours |
| Treatment Vehicle: | RPMI 1640 media (lacking the respective metabolite) |
| Cell Storage: | Temperature and gas composition controlled incubator |
| 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: | SP002112 |
| 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 | AN003294 |
|---|---|
| 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 Q Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | Peak Abundance (normalized, blank subtracted, and corrected for baseline noise) |
Chromatography:
| Chromatography ID: | CH002433 |
| 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; 2.5 uM medronic acid |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003064 |
| Analysis ID: | AN003294 |
| Instrument Name: | Thermo Q 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 |
