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MB Sample ID: SA209668
Local Sample ID: | RF7-09102018-3 |
Subject ID: | SU002267 |
Subject Type: | Cultured cells |
Subject Species: | Plasmodium falciparum |
Taxonomy ID: | 5833 |
Genotype Strain: | DD2B2 |
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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 |
---|---|---|---|---|
RF7-09102018-3 | SA209668 | 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 |