Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA093415

Local Sample ID:	H470.1_t7
Subject ID:	SU001351
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU001351
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
H470.1_t7	SA093415	FL013369	70nM DHA	Treatment

Collection:

Collection ID:	CO001345
Collection Summary:	P. falciparum parasites were cultured at 3% hematocrit in human O+ RBCs (Interstate blood bank, USA) and P. falciparum culture media comprising of RPMI1640 (Thermo Fisher Scientific) supplemented with 0.5% (w/v) Albumax II, 50mg/L hypoxanthine, 0.2% NaHCO3, 25mM HEPES and 10mg/L gentamycin (Fidock et al., 1998). Parasites were cultured at 37ºC in 5% O2, 5%CO2 and 90% N2. For the collection of RNA, proteins and metabolite extracts, parasite cultures at 3% hematocrit and 20 mL or 200 mL volumes were kept in T75 or T225 flasks respectively, with daily media changes. Parasite lines were genotyped by Sanger sequencing for the k13 gene to verify their identities before the start of an experiment.
Sample Type:	Parasite

Treatment:

Treatment ID:	TR001366
Treatment Summary:	Mycoplasma-free Cam3.IIC580Y and Cam3.IIWT parasites were doubly synchronized by 5% D-Sorbitol in each generation for at least two generations. 0-3 hpi early rings of each parasite line were treated for 3h at 350 nM or 70 nM DHA along with vehicle-treated 0.05% DMSO controls in two to three independent experiments. 24 hpi trophozoites were similarly treated with DHA or DMSO control in a single experiment for each parasite line and subsequently magnetically enriched using MACS CS columns on the SuperMACS™ II Separator (Miltenyi Biotec, Inc.) to remove uninfected RBCs.

Treatment ID:

TR001366

Treatment Summary:

Mycoplasma-free Cam3.IIC580Y and Cam3.IIWT parasites were doubly synchronized by 5% D-Sorbitol in each generation for at least two generations. 0-3 hpi early rings of each parasite line were treated for 3h at 350 nM or 70 nM DHA along with vehicle-treated 0.05% DMSO controls in two to three independent experiments. 24 hpi trophozoites were similarly treated with DHA or DMSO control in a single experiment for each parasite line and subsequently magnetically enriched using MACS CS columns on the SuperMACS™ II Separator (Miltenyi Biotec, Inc.) to remove uninfected RBCs.

Sample Preparation:

Sampleprep ID:	SP001359
Sampleprep Summary:	The K13 mutant and WT ring-stage parasites were run in parallel for each metabolomic experiment to allow direct comparisons of their metabolomic states and the effects of DHA. Metabolites from saponin-lysed parasites were extracted with cold methanol along with spike-in control [13C4, 15N1]-Aspartate (Cambridge Isotope) as an internal LC/MS standard to correct for technical variation arising from sample processing in the data analysis phase, as described previously (Allman et al., 2016).Samples were injected on a Thermo Exactive Plus Orbitrap in negative ion mode (Lu, W. et al., Anal. Chem. 2010.).

Sampleprep ID:

SP001359

Sampleprep Summary:

The K13 mutant and WT ring-stage parasites were run in parallel for each metabolomic experiment to allow direct comparisons of their metabolomic states and the effects of DHA. Metabolites from saponin-lysed parasites were extracted with cold methanol along with spike-in control [13C4, 15N1]-Aspartate (Cambridge Isotope) as an internal LC/MS standard to correct for technical variation arising from sample processing in the data analysis phase, as described previously (Allman et al., 2016).Samples were injected on a Thermo Exactive Plus Orbitrap in negative ion mode (Lu, W. et al., Anal. Chem. 2010.).

Combined analysis:

Analysis ID	AN002120
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000
Column	Phenomenex Synergi Hydro RP 100 A (100 x 2mm,2.5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap
Ion Mode	NEGATIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH001553
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Phenomenex Synergi Hydro RP 100 A (100 x 2mm,2.5um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001975
Analysis ID:	AN002120
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data files from the Thermo Exactive Plus orbitrap (.raw) were converted to a format compatible with our analysis software (.raw¡.mzXML) and spectral data (.mzXML files) were visualized in MAVEN. The labeled [13C4, 15N1]-Aspartate internal standard intensity was assessed for technical reproducibility. Peaks for each metabolite in the targeted library were identified based on proximity to standard retention time, the observed mass falling within 10 ppm of the expected m/z (calculated from the monoisotopic mass), and the signal/blank ratio (minimum, 10,000 ions). Based upon the above criteria, peaks were manually inspected and demarcated as good or bad based on peak shape. Peak areas were exported into an R working environment (http://www.R-project.org) to calculate log2 fold changes for each sample compared to an untreated control. Metabolites that were not reliably detected across 90% of all the trials were removed prior to additional analysis to minimize subsequent imputation bias. The peak areas for any remaining metabolites not detected were imputed to have 10,000 ions, and metabolites detected below background levels (negative after blank subtraction) were maintained as “0” prior to averaging and log2 calculation. Because our extraction method did not include a wash step we excluded metabolites found in the RPMI-based medium.
Ion Mode:	NEGATIVE