{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002024","ANALYSIS_ID":"AN003294","VERSION":"1","CREATED_ON":"December 20, 2021, 8:17 am"},

"PROJECT":{"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"},

"STUDY":{"STUDY_TITLE":"Plasmodium falciparum stable-isotope carbon labeling to explore metabolic consequences of keto–acid dehydrogenase disruption","STUDY_SUMMARY":"Plasmodium 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","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","NUM_GROUPS":"16","TOTAL_SUBJECTS":"146"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Plasmodium falciparum","TAXONOMY_ID":"5833","GENOTYPE_STRAIN":"NF54 attB","GENDER":"Not applicable","CELL_COUNTS":"1x10^8"},
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"Sample ID":"20191120-BKE2_glutamine-3",
"Factors":{"Treatment":"20191120-BKE2_glutamine"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-BKE2_glutamine-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-Blank1",
"Factors":{"Treatment":"20191120-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-Blank1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-Blank2",
"Factors":{"Treatment":"20191120-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-Blank2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-Blank3",
"Factors":{"Treatment":"20191120-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-Blank3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-Blank4",
"Factors":{"Treatment":"20191120-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-Blank4.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-Blank5",
"Factors":{"Treatment":"20191120-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-Blank5.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-QC1",
"Factors":{"Treatment":"20191120-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-QC1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-QC2",
"Factors":{"Treatment":"20191120-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-QC2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191120-QC3",
"Factors":{"Treatment":"20191120-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20191120-QC3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-Blank1",
"Factors":{"Treatment":"20191216-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-Blank1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-Blank2",
"Factors":{"Treatment":"20191216-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-Blank2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-Blank3",
"Factors":{"Treatment":"20191216-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-Blank3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-Blank4",
"Factors":{"Treatment":"20191216-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-Blank4.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-Blank5",
"Factors":{"Treatment":"20191216-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-Blank5.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-LipL2-glucose-1",
"Factors":{"Treatment":"20191216-LipL2-glucose"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-LipL2-glucose-1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-LipL2-glucose-2",
"Factors":{"Treatment":"20191216-LipL2-glucose"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-LipL2-glucose-2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-LipL2-glucose-3",
"Factors":{"Treatment":"20191216-LipL2-glucose"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-LipL2-glucose-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-LipL2-glutamine-1",
"Factors":{"Treatment":"20191216-LipL2-glutamine"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-LipL2-glutamine-1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-LipL2-glutamine-2",
"Factors":{"Treatment":"20191216-LipL2-glutamine"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-LipL2-glutamine-2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-LipL2-glutamine-3",
"Factors":{"Treatment":"20191216-LipL2-glutamine"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-LipL2-glutamine-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-QC1",
"Factors":{"Treatment":"20191216-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-QC1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-QC2",
"Factors":{"Treatment":"20191216-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-QC2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-QC3",
"Factors":{"Treatment":"20191216-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-QC3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-WT-glucose-1",
"Factors":{"Treatment":"20191216-WT-glucose"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-WT-glucose-1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-WT-glucose-2",
"Factors":{"Treatment":"20191216-WT-glucose"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-WT-glucose-2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-WT-glucose-3",
"Factors":{"Treatment":"20191216-WT-glucose"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-WT-glucose-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-WT-glutamine-1",
"Factors":{"Treatment":"20191216-WT-glutamine"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-WT-glutamine-1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-WT-glutamine-2",
"Factors":{"Treatment":"20191216-WT-glutamine"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-WT-glutamine-2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20191216-WT-glutamine-3",
"Factors":{"Treatment":"20191216-WT-glutamine"},
"Additional sample data":{"RAW_FILE_NAME":"20191216-WT-glutamine-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glucose-1",
"Factors":{"Treatment":"20200810-BE12-Glucose-B1"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glucose-1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glucose-2",
"Factors":{"Treatment":"20200810-BE12-Glucose-B1"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glucose-2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glucose-3",
"Factors":{"Treatment":"20200810-BE12-Glucose-B1"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glucose-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glucose-4",
"Factors":{"Treatment":"20200810-BE12-Glucose-B2"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glucose-4.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glucose-5",
"Factors":{"Treatment":"20200810-BE12-Glucose-B2"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glucose-5.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glucose-6",
"Factors":{"Treatment":"20200810-BE12-Glucose-B2"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glucose-6.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glutamine-1",
"Factors":{"Treatment":"20200810-BE12-Glutamine-B1"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glutamine-1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glutamine-2",
"Factors":{"Treatment":"20200810-BE12-Glutamine-B1"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glutamine-2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glutamine-3",
"Factors":{"Treatment":"20200810-BE12-Glutamine-B1"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glutamine-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glutamine-4",
"Factors":{"Treatment":"20200810-BE12-Glutamine-B2"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glutamine-4.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glutamine-5",
"Factors":{"Treatment":"20200810-BE12-Glutamine-B2"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glutamine-5.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-BE12-Glutamine-6",
"Factors":{"Treatment":"20200810-BE12-Glutamine-B2"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-BE12-Glutamine-6.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-QC-1",
"Factors":{"Treatment":"20200810-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-QC-1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-QC-2",
"Factors":{"Treatment":"20200810-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-QC-2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-QC-3",
"Factors":{"Treatment":"20200810-QC"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-QC-3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-Blank1",
"Factors":{"Treatment":"20200810-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-Blank1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-Blank2",
"Factors":{"Treatment":"20200810-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-Blank2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-Blank3",
"Factors":{"Treatment":"20200810-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-Blank3.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-Blank4",
"Factors":{"Treatment":"20200810-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-Blank4.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"20200810-Blank5",
"Factors":{"Treatment":"20200810-Blank"},
"Additional sample data":{"RAW_FILE_NAME":"20200810-Blank5.mzXML"}
}
],
"COLLECTION":{"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_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"},

"SAMPLEPREP":{"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"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Ion-pairing method using reverse-phase chromatography setup.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Waters XSelect HSS (2.1 x 100 mm, 2.5 um)","FLOW_RATE":"0.200 mL/minute","COLUMN_TEMPERATURE":"30","SOLVENT_A":"3% methanol, 15 mM acetic acid, 10 mM tributylamine, 2.5 uM medronic acid","SOLVENT_B":"100% methanol"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","OPERATOR_NAME":"Justin Munro"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Data was centroided using MSConvert and converted to .mzXML for utilization in MzMine and El-Maven software."},

"MS_METABOLITE_DATA":{
"Units":"Peak Abundance (normalized, blank subtracted, and corrected for baseline noise)",

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}

}