Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA202334

Local Sample ID:	P_2E7_2
Subject ID:	SU002191
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833

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Subject:

Subject ID:	SU002191
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
P_2E7_2	SA202334	FL024597	iRBC	cell_type
P_2E7_2	SA202334	FL024597	PfAM17-HAglmS-2	treatment

Collection:

Collection ID:	CO002184
Collection Summary:	Heparin synchronized Pf3D7 and PfM17-HAglmS parasites were allowed to invade RBC for 4 h and any remaining schizonts were lysed by sorbitol synchronization. Parasite cultures were then treated for ~ 36 h with 2.5 mM GlcN or for 1 h with 3 at 10x the EC50 (Pf3D7 only) or left untreated. Parasites were harvested at developmentally similar timepoints by centrifugation at 900 g for 5 min and then resuspended in 10 mL of chilled PBS. Parasite metabolism was quenched by cooling samples to between 3-5°C in an ethanol-dry ice bath. The rest of the preparation was performed at 4°C. Parasites were magnet purified on a VarioMACS column and 3x107 parasites were used for downstream analysis. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.
Sample Type:	Blood (whole)

Treatment:

Treatment ID:	TR002203
Treatment Summary:	Heparin synchronized Pf3D7 and PfM17-HAglmS parasites were allowed to invade RBC for 4 h and any remaining schizonts were lysed by sorbitol synchronization. Parasite cultures were then treated for ~ 36 h with 2.5 mM GlcN or for 1 h with 3 at 10x the EC50 (Pf3D7 only) or left untreated. Parasites were harvested at developmentally similar timepoints by centrifugation at 900 g for 5 min and then resuspended in 10 mL of chilled PBS. Parasite metabolism was quenched by cooling samples to between 3-5°C in an ethanol-dry ice bath. The rest of the preparation was performed at 4°C. Parasites were magnet purified on a VarioMACS column and 3x107 parasites were used for downstream analysis. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.

Treatment ID:

TR002203

Treatment Summary:

Heparin synchronized Pf3D7 and PfM17-HAglmS parasites were allowed to invade RBC for 4 h and any remaining schizonts were lysed by sorbitol synchronization. Parasite cultures were then treated for ~ 36 h with 2.5 mM GlcN or for 1 h with 3 at 10x the EC50 (Pf3D7 only) or left untreated. Parasites were harvested at developmentally similar timepoints by centrifugation at 900 g for 5 min and then resuspended in 10 mL of chilled PBS. Parasite metabolism was quenched by cooling samples to between 3-5°C in an ethanol-dry ice bath. The rest of the preparation was performed at 4°C. Parasites were magnet purified on a VarioMACS column and 3x107 parasites were used for downstream analysis. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.

Sample Preparation:

Sampleprep ID:	SP002197
Sampleprep Summary:	Heparin synchronized Pf3D7 and PfM17-HAglmS parasites were allowed to invade RBC for 4 h and any remaining schizonts were lysed by sorbitol synchronization. Parasite cultures were then treated for ~ 36 h with 2.5 mM GlcN or for 1 h with 3 at 10x the EC50 (Pf3D7 only) or left untreated. Parasites were harvested at developmentally similar timepoints by centrifugation at 900 g for 5 min and then resuspended in 10 mL of chilled PBS. Parasite metabolism was quenched by cooling samples to between 3-5°C in an ethanol-dry ice bath. The rest of the preparation was performed at 4°C. Parasites were magnet purified on a VarioMACS column and 3x107 parasites were used for downstream analysis. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.

Sampleprep ID:

SP002197

Sampleprep Summary:

Combined analysis:

Analysis ID	AN003444	AN003445
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) equipped with a guard (SeQuant,Merck)	ZIC-pHILIC (150 x 4.6mm,5um) equipped with a guard (SeQuant,Merck)
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	relative intensity	relative intensity

Chromatography:

Chromatography ID:	CH002545
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	ZIC-pHILIC (150 x 4.6mm,5um) equipped with a guard (SeQuant,Merck)
Chromatography Type:	HILIC

MS:

MS ID:	MS003207
Analysis ID:	AN003444
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. LC MS peak heights representing metabolite abundances were normalised by median peak height. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times.
Ion Mode:	POSITIVE

MS ID:	MS003208
Analysis ID:	AN003445
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. LC MS peak heights representing metabolite abundances were normalised by median peak height. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times.
Ion Mode:	NEGATIVE