Summary of Study ST002024

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001286. The data can be accessed directly via it's Project DOI: 10.21228/M8GQ5C This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002024
Study TitlePlasmodium falciparum stable-isotope carbon labeling to explore metabolic consequences of keto–acid dehydrogenase disruption
Study SummaryPlasmodium falciparum cells in culture were treated with respective universally labelled carbon-13 metabolites for 2.5 hours at standard culture concentrations (glucose or glutamine) or 5 mM (acetate). Metabolites were isolated using 90% methanol, dried, reconstituted in HPLC-grade water, and analyzed by HPLC/MS. Resulting data were analyzed and compiled to generate study data.
Institute
Pennsylvania State University
DepartmentChemistry
LaboratoryLlinás Laboratory
Last NameLlinás
First NameManuel
AddressW126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA
Emailmul27@psu.edu
Phone814-867-3444
Submit Date2021-07-15
Num Groups16
Total Subjects146
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2022-10-03
Release Version1
Manuel Llinás Manuel Llinás
https://dx.doi.org/10.21228/M8GQ5C
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001286
Project DOI:doi: 10.21228/M8GQ5C
Project Title:Plasmodium falciparum stable-isotope carbon labeling to explore metabolic consequences of keto–acid dehydrogenase disruption
Project Summary:Plasmodium falciparum is the most virulent species of parasites that cause malaria. Among other differences, these parasites have a limited reliance on mitochondrial metabolism during their asexual blood stages when compared to model eukaryotes or other parasite stages. Our study aimed to analyze lines of these parasites where one or more of the keto-acid dehydrogenase enzymes that are localized to the mitochondrion were disrupted to determine the importance of acetyl-CoA generation within this organelle. We utilized a targeted HPLC/MS-based approach to profile metabolite abundances and obtain the fractional incorporation of metabolites that are critical to central carbon metabolism (glucose, glutamine, or acetate) into acetyl-CoA and other landmark metabolites across a 2.5-hour time window. Glucose or glutamine were added at standard culture conditions. Acetate experiments used 5 mM acetate, which was shown to permit growth of synthetic lethal lines. The results of these studies demonstrated that synthetic lethal mutant parasites, which were observed when both mitochondrial keto-acid dehydrogenases (PF3D7_0303700.1 and PF3D7_1320800.1) were disrupted, resulted in the lack of incorporation of heavy glucose into acetyl-CoA. However, lines that lacked the synthetic lethal phenotype also resulted in essentially a complete lack of glucose incorporation into acetyl-CoA as long as the mPDH-like subunits (PF3D7_1312600.1 and PF3D7_0303700.1) were disrupted, which suggests that the α-ketoglutarate dehydrogenase enzyme possesses a small amount of enzymatic activity to rescue this metabolic disruption. Furthermore, acetate supplementation experiments have demonstrated that exogenously supplied acetate circumvents these lethal effects and is not notably incorporated into the TCA cycle, which highlights the ability of the nuclear/cytosolic acetyl-CoA synthetase activity to circumvent mitochondrial metabolism given the presence of excess acetate. These studies are necessary for understanding pharmacology effects of relatively new acetyl-CoA-production disrupting drugs and possible resistance mechanisms that may evolve in the future.
Institute:Pennsylvania State University
Department:Chemistry
Laboratory:Llinás Laboratory
Last Name:Llinás
First Name:Manuel
Address:W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA
Email:mul27@psu.edu
Phone:814-867-3444
Funding Source:This work was supported by the Bill and Melinda Gates Foundation (OPP1054480) and the NIH NRSA Integrative Analysis of Metabolic Phenotypes T32 DK120509

Subject:

Subject ID:SU002106
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum
Taxonomy ID:5833
Genotype Strain:NF54 attB
Gender:Not applicable
Cell Counts:1x10^8

Factors:

Subject type: Cultured cells; Subject species: Plasmodium falciparum (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA18959520190304-Blank120190304-Blank
SA18959620190304-Blank420190304-Blank
SA18959720190304-Blank520190304-Blank
SA18959820190304-Blank320190304-Blank
SA18959920190304-Blank220190304-Blank
SA18960020190304-LipL2_acetate320190304-LipL2_AP_acetate
SA18960120190304-LipL2_acetate220190304-LipL2_AP_acetate
SA18960220190304-LipL2_acetate120190304-LipL2_AP_acetate
SA18960320190304-LipL2_glucose320190304-LipL2_AP_glucose
SA18960420190304-LipL2_glucose220190304-LipL2_AP_glucose
SA18960520190304-LipL2_glucose120190304-LipL2_AP_glucose
SA18960620190304-LipL2_glutamine120190304-LipL2_AP_glutamine
SA18960720190304-LipL2_glutamine320190304-LipL2_AP_glutamine
SA18960820190304-LipL2_glutamine220190304-LipL2_AP_glutamine
SA18960920190304-QC320190304-QC
SA18961020190304-QC120190304-QC
SA18961120190304-QC220190304-QC
SA18961220190304-WT_acetate320190304-WT_AP_acetate
SA18961320190304-WT_acetate120190304-WT_AP_acetate
SA18961420190304-WT_acetate220190304-WT_AP_acetate
SA18961520190304-WT_glucose120190304-WT_AP_glucose
SA18961620190304-WT_glucose320190304-WT_AP_glucose
SA18961720190304-WT_glucose220190304-WT_AP_glucose
SA18961820190304-WT_glutamine320190304-WT_AP_glutamine
SA18961920190304-WT_glutamine220190304-WT_AP_glutamine
SA18962020190304-WT_glutamine120190304-WT_AP_glutamine
SA18962120190313-Blank520190313-Blank
SA18962220190313-Blank420190313-Blank
SA18962320190313-Blank220190313-Blank
SA18962420190313-Blank320190313-Blank
SA18962520190313-Blank120190313-Blank
SA18962620190313-LipL2_acetate-320190313-LipL2_acetate
SA18962720190313-LipL2_acetate-220190313-LipL2_acetate
SA18962820190313-LipL2_acetate-120190313-LipL2_acetate
SA18962920190313-LipL2_glucose-320190313-LipL2_glucose
SA18963020190313-LipL2_glucose-220190313-LipL2_glucose
SA18963120190313-LipL2_glucose-120190313-LipL2_glucose
SA18963220190313-LipL2_glutamine-320190313-LipL2_glutamine
SA18963320190313-LipL2_glutamine-220190313-LipL2_glutamine
SA18963420190313-LipL2_glutamine-120190313-LipL2_glutamine
SA18963520190313-QC320190313-QC
SA18963620190313-QC220190313-QC
SA18963720190313-QC120190313-QC
SA18963820190313-WT_acetate-320190313-WT_acetate
SA18963920190313-WT_acetate-120190313-WT_acetate
SA18964020190313-WT_acetate-220190313-WT_acetate
SA18964120190313-WT_glucose-320190313-WT_glucose
SA18964220190313-WT_glucose-220190313-WT_glucose
SA18964320190313-WT_glucose-120190313-WT_glucose
SA18964420190313-WT_glutamine-320190313-WT_glutamine
SA18964520190313-WT_glutamine-120190313-WT_glutamine
SA18964620190313-WT_glutamine-220190313-WT_glutamine
SA18964720190401-Blank520190401-Blank
SA18964820190401-Blank420190401-Blank
SA18964920190401-Blank320190401-Blank
SA18965020190401-Blank220190401-Blank
SA18965120190401-Blank120190401-Blank
SA18965220190401-LipL2-glucose-320190401-LipL2-glucose
SA18965320190401-LipL2-glucose-220190401-LipL2-glucose
SA18965420190401-LipL2-glucose-120190401-LipL2-glucose
SA18965520190401-LipL2-glutamine-320190401-LipL2-glutamine
SA18965620190401-LipL2-glutamine-120190401-LipL2-glutamine
SA18965720190401-LipL2-glutamine-220190401-LipL2-glutamine
SA18965820190401-QC320190401-QC
SA18965920190401-QC220190401-QC
SA18966020190401-QC120190401-QC
SA18966120190401-WT-glucose-120190401-WT-glucose
SA18966220190401-WT-glucose-320190401-WT-glucose
SA18966320190401-WT-glucose-220190401-WT-glucose
SA18966420190401-WT-glutamine-320190401-WT-glutamine
SA18966520190401-WT-glutamine-120190401-WT-glutamine
SA18966620190401-WT-glutamine-220190401-WT-glutamine
SA18966720191107-BKE2_glucose-320191107-BKE2_glucose
SA18966820191107-BKE2_glucose-220191107-BKE2_glucose
SA18966920191107-BKE2_glucose-120191107-BKE2_glucose
SA18967020191107-BKE2_glutamine-120191107-BKE2_glutamine
SA18967120191107-BKE2_glutamine-320191107-BKE2_glutamine
SA18967220191107-BKE2_glutamine-220191107-BKE2_glutamine
SA18967320191107-Blank420191107-Blank
SA18967420191107-Blank520191107-Blank
SA18967520191107-Blank120191107-Blank
SA18967620191107-Blank220191107-Blank
SA18967720191107-Blank320191107-Blank
SA18967820191107-QC320191107-QC
SA18967920191107-QC220191107-QC
SA18968020191107-QC120191107-QC
SA18968120191107-WT_glucose-320191107-WT_glucose
SA18968220191107-WT_glucose-220191107-WT_glucose
SA18968320191107-WT_glucose-120191107-WT_glucose
SA18968420191107-WT_glutamine-320191107-WT_glutamine
SA18968520191107-WT_glutamine-220191107-WT_glutamine
SA18968620191107-WT_glutamine-120191107-WT_glutamine
SA18968720191120-BKE2_glucose-320191120-BKE2_glucose
SA18968820191120-BKE2_glucose-220191120-BKE2_glucose
SA18968920191120-BKE2_glucose-120191120-BKE2_glucose
SA18969020191120-BKE2_glutamine-320191120-BKE2_glutamine
SA18969120191120-BKE2_glutamine-120191120-BKE2_glutamine
SA18969220191120-BKE2_glutamine-220191120-BKE2_glutamine
SA18969320191120-Blank520191120-Blank
SA18969420191120-Blank420191120-Blank
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Collection:

Collection ID:CO002099
Collection Summary:Plasmodium falciparum NF54 attB parasites containing different genetic backgrounds were cultured in RPMI 1640 medium and magnetically enriched to increase the infected to uninfected RBC ratio. Following hemocytometer counts, 1x10^8 parasites were measured per condition into 5 mL of total media for 2.5 hours in the presence of the universally labelled carbon-13 metabolite (glucose, glutamine, or acetate). Medium was aspirated until 1 mL remained on each sample, the sample was transferred to a micro-centrifuge tube, spun, and the medium was aspirated.
Collection Protocol Filename:Metabolite_Extraction_for_LCMS_2017.pdf
Sample Type:Cultured cells
Collection Location:Millenium Science Complex, University Park, Pennsylvania
Storage Conditions:-80℃
Collection Vials:1.5 mL eppendorf
Storage Vials:1.5 mL eppendorf
Collection Tube Temp:On ice
Tissue Cell Quantity Taken:1x10^8 cells per sample in 1 mL total volume

Treatment:

Treatment ID:TR002118
Treatment Summary:Plasmodium falciparum NF54 attB parasites were cultured in RPMI 1640 medium and magnetically enriched to increase the infected to uninfected RBC ratio. Following hemocytometer counts, 1x10^8 parasites were measured per condition into 5 mL of total media for 2.5 hours in the presence of the carbon-13 metabolite (glucose, glutamine, or acetate). Wild-type or WT represents control parasites without additional alterations. Blanks are sample tubes that follow the same procedures as samples following the quenching of metabolism. Both Pool and QC samples are combined samples of all samples from the analytical batch on that particular day. Dates in YYYYMMDD format are appended to individual samples to indicate the batch in which they were processed.
Treatment Protocol Filename:Metabolite_Extraction_for_LCMS_2017.pdf
Treatment Compound:Universally labelled glucose, glutamine, or acetate
Treatment Route:Transfer to media by pipette
Treatment Dosevolume:50 uL (glucose), 25 uL (glutamine), or 10 uL (acetate)
Treatment Doseduration:2.5 hours
Treatment Vehicle:RPMI 1640 media (lacking the respective metabolite)
Cell Storage:Temperature and gas composition controlled incubator
Cell Growth Container:6-well plate
Cell Growth Config:5 mL in each sample well, 1x10^8 infected red blood cells per well, conditions performed in triplicate
Cell Media:RPMI 1640 containing Albumax, Gentamycin, Hypoxanthine, HEPES, Sodium Bicarbonate

Sample Preparation:

Sampleprep ID:SP002112
Sampleprep Summary:Once samples were obtained, metabolism was quenched with 90% methanol containing 0.25 uM labeled aspartate, cells were centrifuged, and the supernatant was removed by nitrogen drying. Sample was reconstituted in 3% HPLC-grade methanol and run on the instrument.
Processing Method:Wash, spin, quench, spin, dry, store at -80 C until run on the instrument, resuspend
Processing Storage Conditions:On ice
Extraction Method:90% methanol extraction
Extract Cleanup:Centrifugation and nitrogen drying
Sample Resuspension:100 uL 3% methanol with 1 uM chlorpropamide
Sample Spiking:0.25 uM Labelled Aspartate, 1 uM chlorpropamide

Combined analysis:

Analysis ID AN003294
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Dionex Ultimate 3000
Column Waters XSelect HSS (100 x 2.1mm,2.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode NEGATIVE
Units Peak Abundance (normalized, blank subtracted, and corrected for baseline noise)

Chromatography:

Chromatography ID:CH002433
Chromatography Summary:Ion-pairing method using reverse-phase chromatography setup.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters XSelect HSS (100 x 2.1mm,2.5um)
Column Temperature:30
Flow Rate:0.200 mL/minute
Solvent A:97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine; 2.5 uM medronic acid
Solvent B:100% methanol
Chromatography Type:Reversed phase

MS:

MS ID:MS003064
Analysis ID:AN003294
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Data was centroided using MSConvert and converted to .mzXML for utilization in MzMine and El-Maven software.
Ion Mode:NEGATIVE
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