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MB Sample ID: SA209676
| Local Sample ID: | Dd2Dd2crtM343L-10092018-2 |
| Subject ID: | SU002267 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | DD2B2 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002267 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Genotype Strain: | DD2B2 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Dd2Dd2crtM343L-10092018-2 | SA209676 | FL025590 | Dd2+M343L | PfCRT Haplotype |
Collection:
| Collection ID: | CO002260 |
| Collection Summary: | Mycoplasma-free parasites were sorbitol-synchronized in each generation for at least two generations followed by magnetic enrichment of 32 hr post-invasion trophozoites using MACS CS columns on a SuperMACS™ II Separator (Miltenyi Biotec, Inc.) to remove uninfected RBCs. Trophozoite counts were determined on a hemocytometer. Parasites were lysed in 1 mL of 90% cold methanol, containing 0.5 μM of the internal standard [13C4, 15N1]-Aspartate (Cambridge Isotope) to correct for technical variations arising from sample processing. Samples were vortexed to disrupt cell pellets and to generate uniform homogenates, which were centrifuged (13,000´ g for 10 min) and the supernatants then harvested. Supernatants were dried under nitrogen prior to resuspension in HPLC-grade water for LC-MS analysis. |
| Sample Type: | Plasmodium cells |
Treatment:
| Treatment ID: | TR002279 |
| Treatment Summary: | Cells were all untreated, but had different genetic backgrounds. |
Sample Preparation:
| Sampleprep ID: | SP002273 |
| Sampleprep Summary: | Supernatants were dried under nitrogen prior to resuspension in HPLC-grade water for LC-MS analysis. |
| Processing Method: | Lysis |
| Processing Storage Conditions: | 4℃ |
| Extraction Method: | See SA Cobbold et al JBC 2013 |
| Extract Cleanup: | Clarification via centrifugation followed by nitrogen drying |
| Extract Storage: | -80℃ |
| Sample Resuspension: | HPLC-grade water spiked with chlorpropamide |
| Sample Spiking: | Internal Standard 13C4, 15N1-Aspartate used for sample preparation normalizer |
Combined analysis:
| Analysis ID | AN003572 | AN003573 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Prominence 20 UFLCXR | Prominence 20 UFLCXR |
| Column | Waters Acquity BEH C18 (100 x 2mm,1.7um) | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | ABI Sciex 5600 TripleTOF | ABI Sciex 5600 TripleTOF |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002641 |
| Chromatography Summary: | Samples (5ul) were separated by reverse phase HPLC using a Prominence 20 UFLCXR system (Shimadzu, Columbia, MD) with a Waters (Milford, MA) BEH C18 column (100mm x 2.1mm 1.7 um particle size) maintained at 55C and a 20 minute aqueous acetonitrile gradient, at a flow rate of 250 ul/min. Solvent A was HPLC grade water with 0.1% formic acid and Solvent B was HPLC grade acetonitrile with 0.1% formic acid. The initial condition were 97% A and 3 % B, increasing to 45% B at 10 min, 75% B at 12 min where it was held at 75% B until 17.5 min before returning to the initial conditions. |
| Instrument Name: | Prominence 20 UFLCXR |
| Column Name: | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
| Column Temperature: | 55 |
| Flow Gradient: | The initial conditions were 97% A and 3 % B, increasing to 45% B at 10 min, 75% B at 12 min where it was held at 75% B until 17.5 min before returning to the initial conditions. |
| Flow Rate: | 250 ul/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003329 |
| Analysis ID: | AN003572 |
| Instrument Name: | ABI Sciex 5600 TripleTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The eluate was delivered into a 5600 (QTOF) TripleTOF using a Duospray™ ion source (all Sciex, Framingham, MA).The capillary voltage was set at 5.5 kV in positive/negative ion mode with a declustering potential of 80V. The mass spectrometer was operated with a 100 ms TOF scan from 50 to 1000 m/z, and 16 MS/MS product ion scans (100 ms) per duty cycle using a collision energy of 50V with a 20V spread. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003330 |
| Analysis ID: | AN003573 |
| Instrument Name: | ABI Sciex 5600 TripleTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The eluate was delivered into a 5600 (QTOF) TripleTOF using a Duospray™ ion source (all Sciex, Framingham, MA).The capillary voltage was set at 5.5 kV in positive/negative ion mode with a declustering potential of 80V. The mass spectrometer was operated with a 100 ms TOF scan from 50 to 1000 m/z, and 16 MS/MS product ion scans (100 ms) per duty cycle using a collision energy of 50V with a 20V spread. |
| Ion Mode: | POSITIVE |
