Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA081526

Local Sample ID:	P_Atov_3
Subject ID:	SU001241
Subject Type:	Other
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833

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Subject:

Subject ID:	SU001241
Subject Type:	Other
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
P_Atov_3	SA081526	FL012430	Atov treated	Condition

Collection:

Collection ID:	CO001235
Collection Summary:	Plasmodium falciparum infected red blood cells treated with different drugs were counted washed and metabolites extracted used 100% methanol
Sample Type:	Plasmodium cells

Treatment:

Treatment ID:	TR001256
Treatment Summary:	The metabolism of mid-trophozoite stage parasites (24-28 h post invasion) in response to treatment with a panel of antimalarial compounds was investigated. Cultures (200 μl) were adjusted to 8% parasitemia and 2% hematocrit before being exposed to test compounds; JPC-3210, AQm, PYN, ATQ, DHA, CQ, MQ, LF and TQ (1 μM) for 1 h in a flat bottom 96 well plate. At least three replicates of each compound and seven replicates of an untreated control which contained an equivalent volume of DMSO vehicle (0.01% final concentration) were analysed.

Treatment ID:

TR001256

Treatment Summary:

The metabolism of mid-trophozoite stage parasites (24-28 h post invasion) in response to treatment with a panel of antimalarial compounds was investigated. Cultures (200 μl) were adjusted to 8% parasitemia and 2% hematocrit before being exposed to test compounds; JPC-3210, AQm, PYN, ATQ, DHA, CQ, MQ, LF and TQ (1 μM) for 1 h in a flat bottom 96 well plate. At least three replicates of each compound and seven replicates of an untreated control which contained an equivalent volume of DMSO vehicle (0.01% final concentration) were analysed.

Sample Preparation:

Sampleprep ID:	SP001249
Sampleprep Summary:	Following drug incubation, metabolites were extracted as detailed. In short, after incubation with drug, culture medium was aspirated from each well, and the metabolism of infected red blood cells (iRBCs) was quenched by the addition of ice-cold phosphate-buffered saline (PBS). Cells were pelleted by centrifugation for 5 min at 1,000 × g, and the PBS supernatant was removed prior to the addition of 135 μL ice cold methanol (containing the internal standard compounds; CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS [N-cyclohexyl-3-aminopropane-sulfonic acid], and PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)]). Samples were rapidly mixed by pipetting three times to extract iRBC metabolites. Samples were left on ice with gentle agitation for 60 min and then centrifuged at 3,000 × g to remove insoluble material. Supernatants were transferred to glass high-performance liquid chromatography (HPLC) vials and stored at −80°C until analysis. An aliquot (10 μL) of each sample was combined to generate a pooled biological quality control (PBQC) sample, which was used to monitor downstream sample stability and analytical reproducibility.

Sampleprep ID:

SP001249

Sampleprep Summary:

Following drug incubation, metabolites were extracted as detailed. In short, after incubation with drug, culture medium was aspirated from each well, and the metabolism of infected red blood cells (iRBCs) was quenched by the addition of ice-cold phosphate-buffered saline (PBS). Cells were pelleted by centrifugation for 5 min at 1,000 × g, and the PBS supernatant was removed prior to the addition of 135 μL ice cold methanol (containing the internal standard compounds; CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS [N-cyclohexyl-3-aminopropane-sulfonic acid], and PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)]). Samples were rapidly mixed by pipetting three times to extract iRBC metabolites. Samples were left on ice with gentle agitation for 60 min and then centrifuged at 3,000 × g to remove insoluble material. Supernatants were transferred to glass high-performance liquid chromatography (HPLC) vials and stored at −80°C until analysis. An aliquot (10 μL) of each sample was combined to generate a pooled biological quality control (PBQC) sample, which was used to monitor downstream sample stability and analytical reproducibility.

Combined analysis:

Analysis ID	AN001950	AN001951
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000
Column	ZIC-pHILIC (150 x 4.6mm,5um)	ZIC-pHILIC (150 x 4.6mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Signal Intensity	Signal Intensity

Chromatography:

Chromatography ID:	CH001414
Chromatography Summary:	Briefly, samples (10 μL) were injected onto a Dionex RSLC U3000 LC system (Thermo) fitted with a ZIC-pHILIC column (5 μm particle size, 4.6 by 150 mm; Merck) and 20 mM ammonium carbonate (A) and acetonitrile (B) were used as the mobile phases. A 30 min gradient starting from 80% B to 40% B over 20 min, followed by washing at 5% B for 3 min and re-equilibration at 80% B, was used.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	ZIC-pHILIC (150 x 4.6mm,5um)
Flow Gradient:	A 30 min gradient starting from 80% B to 40% B over 20 min, followed by washing at 5% B for 3 min and re-equilibration at 80% B, was used.
Solvent A:	100% water; 20 mM ammonium carbonate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS001805
Analysis ID:	AN001950
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	-
Ion Mode:	POSITIVE

MS ID:	MS001806
Analysis ID:	AN001951
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	-
Ion Mode:	NEGATIVE