Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA069188

Local Sample ID:	P_397264_1
Subject ID:	SU001072
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833
Genotype Strain:	3D7
Subject Comments:	Malaria parasite

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Subject:

Subject ID:	SU001072
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833
Genotype Strain:	3D7
Subject Comments:	Malaria parasite

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
P_397264_1	SA069188	FL011039	MMV397264	drug treatment

Collection:

Collection ID:	CO001066
Collection Summary:	Plasmodium falciparum (3D7 strain) parasites were cultured in vitro according to the established method (Trager and Jensen 1976, DOI: 10.1126/science.781840) with minor modifications and incubated with test compounds as previously described (Creek et al 2016, DOI: 10.1128/AAC.01226-16). Briefly, parasites were brought to a tightly synchronous life stage population (within 4 hours of the 48 hour life cycle) by treating with 5% (w/v) sorbitol twice at an interval of 14 hours, and incubated for a further 58 hours to bring all parasites to mid-trophozoite stage (27-31 hours post infection). These cells were used for drug treatment and further analysis.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR001086
Treatment Summary:	In 96 well plates, 200 μl cultures at 7% parasitemia and 3% haematocrit were incubated with 1 µM of test compounds for a further 5 hours (32-36 hours post infection). Each compound was incubated in four replicates and untreated controls were treated with DMSO.

Sample Preparation:

Sampleprep ID:	SP001079
Sampleprep Summary:	After incubation with the test compounds for 5 hours, all red blood cells were settled at the bottom of the culture wells. Culture medium was carefully removed and the metabolism of the cells was quenched by placing the plate on ice and adding ice-cold phosphate buffered saline (PBS) to the culture wells. All subsequent extraction steps were performed on ice. Cells were centrifuged for 5 min at 400g in a chilled centrifuge at 4°C. The supernatant was carefully removed and 135 µl of ice-cold methanol (containing internal standards TRIS, CHAPS, CAPS and PIPES) was added followed by rapid mixing of the cell suspension using the pipette three times. Spent media samples were also prepared by adding 10 µl of the culture supernatants to 140 µl of ice-cold methanol (containing internal standards). All samples were agitated on ice for 1 hour and then centrifuged for 10 min at 1000g in a chilled centrifuge at 4°C. The supernatant was transferred to glass vials and stored at -80°C until analysis.

Sampleprep ID:

SP001079

Sampleprep Summary:

After incubation with the test compounds for 5 hours, all red blood cells were settled at the bottom of the culture wells. Culture medium was carefully removed and the metabolism of the cells was quenched by placing the plate on ice and adding ice-cold phosphate buffered saline (PBS) to the culture wells. All subsequent extraction steps were performed on ice. Cells were centrifuged for 5 min at 400g in a chilled centrifuge at 4°C. The supernatant was carefully removed and 135 µl of ice-cold methanol (containing internal standards TRIS, CHAPS, CAPS and PIPES) was added followed by rapid mixing of the cell suspension using the pipette three times. Spent media samples were also prepared by adding 10 µl of the culture supernatants to 140 µl of ice-cold methanol (containing internal standards). All samples were agitated on ice for 1 hour and then centrifuged for 10 min at 1000g in a chilled centrifuge at 4°C. The supernatant was transferred to glass vials and stored at -80°C until analysis.

Combined analysis:

Analysis ID	AN001694
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Dionex Ultimate 3000 RS
Column	ZIC-pHILIC (Merck Sequant)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	Peak height

Chromatography:

Chromatography ID:	CH001193
Chromatography Summary:	Metabolite extracts were analysed using hydrophilic interaction (HILIC) liquid chromatography (LC) and high resolution mass spectrometry on an Orbitrap system.
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	ZIC-pHILIC (Merck Sequant)
Column Temperature:	25°C
Flow Gradient:	linear gradient -time, %B as follows: 0min- 80%, 15min- 50%, 18min- 5%, 21min- 5%, 24min- 80%, 32min- 80%.
Flow Rate:	300 μL/min
Internal Standard:	internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM)
Sample Injection:	10 μL
Solvent A:	100% water; 20 mM ammonium carbonate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS001569
Analysis ID:	AN001694
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
Ion Mode:	UNSPECIFIED
Capillary Temperature:	300°C
Capillary Voltage:	+50 V
Spray Voltage:	4kV
Resolution Setting:	35000
Scan Range Moverz:	85- 1275 m/z