Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA345856

Local Sample ID:	WJM992_3
Subject ID:	SU003298
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833

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Subject:

Subject ID:	SU003298
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
WJM992_3	SA345856	FL040593	Plasmodium cells	Sample source
WJM992_3	SA345856	FL040593	WJM992	Treatment

Collection:

Collection ID:	CO003291
Collection Summary:	Samples were collected from P. falciparum cultures (3D7 strain) synchronized to the mid trophozoite stage (28-36 hours post invasion). They were magnet purified to achieve a parasitemia of >90% and hematocrit of 0.5%. The culture medium was refreshed immediately before compound incubation.
Sample Type:	Plasmodium cells

Treatment:

Treatment ID:	TR003307
Treatment Summary:	Infected red blood cells were treated with 70 nM of compound 10ah (5 x EC50), 5 nM (5 x EC50), or 20 nM (20 x EC50) of the known PfATP4 inhibitor KAE609 1, or an equivalent volume of vehicle (DMSO) for 5 h with a minimum of three independent incubations per condition.

Sample Preparation:

Sampleprep ID:	SP003305
Sampleprep Summary:	Following compound incubation, cultures were centrifuged at 1,200 g for 3 min, the media was removed, and the cell pellets were washed in 1 mL of ice-cold PBS. Samples were again centrifuged at 1,200 g for 3 min to remove all of the PBS and metabolites were extracted from 5 x 107 cells using 90 µL of ice-cold methanol extraction solvent. Samples were then incubated on an automatic vortex mixer at 4 °C for 1 h before being centrifuged at 21,000 g for 10 min. The supernatants were transferred into high-performance liquid chromatography (HPLC) vials and stored at -80 °C until liquid chromatography-mass spectrometry (LC-MS) analysis. A 10 µL aliquot from each sample was pooled to serve as a quality control sample for monitoring instrument reproducibility and to aid downstream metabolite identification.

Sampleprep ID:

SP003305

Sampleprep Summary:

Following compound incubation, cultures were centrifuged at 1,200 g for 3 min, the media was removed, and the cell pellets were washed in 1 mL of ice-cold PBS. Samples were again centrifuged at 1,200 g for 3 min to remove all of the PBS and metabolites were extracted from 5 x 107 cells using 90 µL of ice-cold methanol extraction solvent. Samples were then incubated on an automatic vortex mixer at 4 °C for 1 h before being centrifuged at 21,000 g for 10 min. The supernatants were transferred into high-performance liquid chromatography (HPLC) vials and stored at -80 °C until liquid chromatography-mass spectrometry (LC-MS) analysis. A 10 µL aliquot from each sample was pooled to serve as a quality control sample for monitoring instrument reproducibility and to aid downstream metabolite identification.

Combined analysis:

Analysis ID	AN005221	AN005222
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Merck SeQuant ZIC-pHILIC (150 x 4.6mm,5um)	Merck SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Orbitrap Exploris 120	Thermo Orbitrap Exploris 120
Ion Mode	POSITIVE	NEGATIVE
Units	Peak height	Peak height

Chromatography:

Chromatography ID:	CH003949
Instrument Name:	Thermo Vanquish
Column Name:	Merck SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
Column Temperature:	25
Flow Gradient:	0–10 min, 80–50% B; 10–12 min, 50–5% B; 12–14 min, 5% B; 14–16 min, 5–80% B and 16–22 min, 80% B
Flow Rate:	0.35 mL/min
Solvent A:	100% Water; 20 mM ammonium carbonate
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004954
Analysis ID:	AN005221
Instrument Name:	Thermo Orbitrap Exploris 120
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data were acquired as a full scan in positive and negative ionization modes with a heated electrospray source and an Orbitrap resolution of 120,000 from 70 to 1,050 m/z. Ion source voltage was 3,500 V in positive mode and 2,500 V in negative mode. The ion transfer tube temperature was 325 °C and the vaporizer temperature was 350 °C. Gas mode was set to static with sheath gas, aux gas, and sweep gas at 50, 10, and 1, respectively. Samples within the LC-MS batch were sorted according to blocks of replicates and randomized. To facilitate metabolite identification, approximately 350 authentic metabolite standards were analyzed before the LC-MS batch, and their peaks and retention time were manually checked using the MZmine software. Pooled biological quality control samples and extraction solvent blanks were analyzed periodically throughout the batch to monitor LC-MS signal reproducibility and assist metabolite identification procedures. Raw LC-MS metabolomics data were analysed using the open source software, IDEOM (http://mzmatch.sourceforge.net/ideom.php). Briefly, the IDEOM workflow uses msconvert to convert raw files to mzXML format, XCMS (Centwave) to pick LC-MS peak signals, and MZmatch for alignment and annotation of related metabolite peaks. Default IDEOM parameters were used to eliminate unwanted noise and artifact peaks. Confident metabolite identification was made by matching accurate masses to the retention time of the ~350 authentic standards. When these authentic standards were unavailable, putative metabolite identification used accurate mass and predicted retention times, as previously described. Metabolite abundance was represented by LC-MS peak height.
Ion Mode:	POSITIVE

MS ID:	MS004955
Analysis ID:	AN005222
Instrument Name:	Thermo Orbitrap Exploris 120
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data were acquired as a full scan in positive and negative ionization modes with a heated electrospray source and an Orbitrap resolution of 120,000 from 70 to 1,050 m/z. Ion source voltage was 3,500 V in positive mode and 2,500 V in negative mode. The ion transfer tube temperature was 325 °C and the vaporizer temperature was 350 °C. Gas mode was set to static with sheath gas, aux gas, and sweep gas at 50, 10, and 1, respectively. Samples within the LC-MS batch were sorted according to blocks of replicates and randomized. To facilitate metabolite identification, approximately 350 authentic metabolite standards were analyzed before the LC-MS batch, and their peaks and retention time were manually checked using the MZmine software. Pooled biological quality control samples and extraction solvent blanks were analyzed periodically throughout the batch to monitor LC-MS signal reproducibility and assist metabolite identification procedures. Raw LC-MS metabolomics data were analysed using the open source software, IDEOM (http://mzmatch.sourceforge.net/ideom.php). Briefly, the IDEOM workflow uses msconvert to convert raw files to mzXML format, XCMS (Centwave) to pick LC-MS peak signals, and MZmatch for alignment and annotation of related metabolite peaks. Default IDEOM parameters were used to eliminate unwanted noise and artifact peaks. Confident metabolite identification was made by matching accurate masses to the retention time of the ~350 authentic standards. When these authentic standards were unavailable, putative metabolite identification used accurate mass and predicted retention times, as previously described. Metabolite abundance was represented by LC-MS peak height.
Ion Mode:	NEGATIVE