#METABOLOMICS WORKBENCH d_shanmugam_20170722_191429 DATATRACK_ID:1169 STUDY_ID:ST000817 ANALYSIS_ID:AN001295 PROJECT_ID:PR000582 VERSION 1 CREATED_ON July 31, 2017, 4:47 pm #PROJECT PR:PROJECT_TITLE Toxoplasma metabolomics PR:PROJECT_TYPE Metabolic alterations in wild type vs glycolytic and gluconeogenesis mutants PR:PROJECT_SUMMARY The focus of this study was to profile the metabolic fates of glucose and PR:PROJECT_SUMMARY glutamine in wild type, glycolytic mutant (hexokinase KO) and gluconeogenesis PR:PROJECT_SUMMARY mutant (PEPCK1 KO) tachyzoite stage T. gondii parasites. 13C6-glucose and PR:PROJECT_SUMMARY 13C5-15N1-glutamine were used as metabolic tracers. Metabolites from glycolysis, PR:PROJECT_SUMMARY pentosephosphate pathway and Kerbs cycle were identified and their isotopomer PR:PROJECT_SUMMARY abundance was quantified in order to visualize metabolic flux across the PR:PROJECT_SUMMARY indicated pathways. A time course (from 0 minutes to 120 minutes) analysis was PR:PROJECT_SUMMARY carried out in the labeling experiments. Here, host cell free extracellular PR:PROJECT_SUMMARY tachyzoite stage T. gondii parasites were used. PR:INSTITUTE CSIR-National Chemical Laboratory PR:DEPARTMENT Biochemical Sciences Division PR:LABORATORY Molecular Parasitology Laboratory PR:LAST_NAME Dhanasekaran PR:FIRST_NAME Shanmugam PR:ADDRESS Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India PR:EMAIL d.shanmugam@ncl.res.in PR:PHONE +91-20-25902719 #STUDY ST:STUDY_TITLE Dynamics of metabolism, proliferation and differentiation in Toxoplasma gondii ST:STUDY_TITLE mutants deficient in glycolysis and gluconeogenesis ST:STUDY_TYPE Time course 13C labeling of Toxoplasma gondii using glucose and glutamine ST:STUDY_SUMMARY The focus of this study was to profile the metabolic fates of glucose and ST:STUDY_SUMMARY glutamine in wild type, glycolytic mutant (hexokinase KO) and gluconeogenesis ST:STUDY_SUMMARY mutant (PEPCK1 KO) tachyzoite stage T. gondii parasites. 13C6-glucose and ST:STUDY_SUMMARY 13C5-15N1-glutamine were used as metabolic tracers. Metabolites from glycolysis, ST:STUDY_SUMMARY pentosephosphate pathway and Kerbs cycle were identified and their isotopomer ST:STUDY_SUMMARY abundance was quantified in order to visualize metabolic flux across the ST:STUDY_SUMMARY indicated pathways. A time course (from 0 minutes to 120 minutes) analysis was ST:STUDY_SUMMARY carried out in the labeling experiments. Here, host cell free extracellular ST:STUDY_SUMMARY tachyzoite stage T. gondii parasites were used. ST:INSTITUTE CSIR-National Chemical Laboratory ST:DEPARTMENT Biochemical Sciences Division ST:LABORATORY Molecular Parasitology Laboratory ST:LAST_NAME Shanmugam ST:FIRST_NAME Dhanasekaran ST:ADDRESS Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India ST:EMAIL d.shanmugam@ncl.res.in ST:PHONE +91-20-25902719 #SUBJECT SU:SUBJECT_TYPE Cells (protozoan parasite) SU:SUBJECT_SPECIES Toxoplasma gondii SU:TAXONOMY_ID 5811 SU:GENOTYPE_STRAIN RH (Type I strain) SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:SUBJECT_COMMENTS Asexual stage (Tachyzoite) SU:CELL_PASSAGE_NUMBER In continuous culture SU:CELL_COUNTS 10^8 cells per sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t000_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t000_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t005_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t005_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t010_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t010_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t030_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t030_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t060_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t060_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t120_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglucose_t120_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t000_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t000_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t005_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t005_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t010_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t010_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t030_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t030_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t060_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t060_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t120_A Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_RH_13Cglutamine_t120_B Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t000_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t000_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t005_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t005_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t010_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t010_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t030_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t030_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t060_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t060_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t120_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglucose_t120_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t000_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t000_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t005_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t005_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t010_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t010_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t030_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t030_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t060_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t060_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t120_A Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_HKKO_13Cglutamine_t120_B Cell line:T. gondii RH HKKO SUBJECT_SAMPLE_FACTORS - Tgon_PEPCKwt_minusglucose_13Cglutamine_1 Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_PEPCKwt_minusglucose_13Cglutamine_2 Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_PEPCKwt_minusglucose_13Cglutamine_3 Cell line:T. gondii RH wt SUBJECT_SAMPLE_FACTORS - Tgon_PEPCKKO_minusglucose_13Cglutamine_4 Cell line:T. gondii RH PEPCKKO SUBJECT_SAMPLE_FACTORS - Tgon_PEPCKKO_minusglucose_13Cglutamine_5 Cell line:T. gondii RH PEPCKKO SUBJECT_SAMPLE_FACTORS - Tgon_PEPCKKO_minusglucose_13Cglutamine_6 Cell line:T. gondii RH PEPCKKO #COLLECTION CO:COLLECTION_SUMMARY Extracellular tachyzoite stage Toxoplasma gondii parasites incubated in either CO:COLLECTION_SUMMARY +13C6-glucose or +13C5-15N2-glutamine containing DMEM medium for 0, 5, 10, 30, CO:COLLECTION_SUMMARY 60 & 120 minutes were collected by centrifugation and immediately extracted with CO:COLLECTION_SUMMARY chilled acetonitrile:water (80:20) mixture and further process for LC-MS CO:COLLECTION_SUMMARY analysis. CO:SAMPLE_TYPE Cells CO:COLLECTION_METHOD Centrifuging cell suspension at 5000 rpm for 5 min at 4 deg. Cel. CO:COLLECTION_TIME At 0, 5, 10, 30, 60 & 120 minutes post labeling with either +13C6-glucose or CO:COLLECTION_TIME +13C5-15N2-glutamine. CO:VOLUMEORAMOUNT_COLLECTED 10^8 cells per sample CO:STORAGE_CONDITIONS -80 deg. Cel. Freezer post extraction until LC-MS analysis can be carried out. CO:COLLECTION_VIALS 1.5 ml capped tubes CO:STORAGE_VIALS 1.5 ml capped tubes CO:COLLECTION_TUBE_TEMP 4 deg. Cel. #TREATMENT TR:TREATMENT_SUMMARY Tachyzoite stage Toxoplasma gondii parasites were freshly isolated from infected TR:TREATMENT_SUMMARY Human Foreskin Fibroblast monolayers and the extracellular parasites were TR:TREATMENT_SUMMARY incubated in DMEM medium supplemented with either +13C6-glucose or TR:TREATMENT_SUMMARY +13C5-15N2-glutamine. Treatment with labeled substrates was done for 0, 5, 10, TR:TREATMENT_SUMMARY 30, 60 & 120 minutes before harvesting and processing the cells for LC-MS TR:TREATMENT_SUMMARY analysis. TR:CELL_STORAGE Liquid nitrogen cell storage system TR:CELL_GROWTH_CONTAINER Standard incubator maintaining 37 deg. Cel. & 5% CO2 TR:CELL_MEDIA DMEM minus glucose medium (cat. No. 11966; Gibco, ThermoFisher Scientific) TR:CELL_MEDIA supplemented with +13C6-glucose. DMEM minus glutamine medium (cat. No. 11960; TR:CELL_MEDIA Gibco, ThermoFisher Scientific) supplemented with +13C5-15N2-glutamine. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolic labeling, metabolite extraction, and LC-MS profiling – DMEM minus SP:SAMPLEPREP_SUMMARY glucose media supplemented with +613C glucose (5.5 mM) and MEM minus glutamine SP:SAMPLEPREP_SUMMARY media supplemented with +513C-+213N glutamine (4 mM) were used as culture SP:SAMPLEPREP_SUMMARY mediums for metabolic labeling studies using RH wt, RH ∆ku80, RH ∆hk, and RH SP:SAMPLEPREP_SUMMARY ∆pepck1 parasites. Freshly isolated extracellular (host cell free) tachyzoites SP:SAMPLEPREP_SUMMARY stage parasites were washed once in complete medium and resuspended in either SP:SAMPLEPREP_SUMMARY 13C labeled glucose or glutamine (Cambridge Isotope Laboratories, Inc.) SP:SAMPLEPREP_SUMMARY containing medium at a density of 108 parasites per milliliter. Labeling was SP:SAMPLEPREP_SUMMARY allowed to proceed over a time period of 5, 10, 15, 30, 60 and 120 minutes in 1 SP:SAMPLEPREP_SUMMARY ml culture suspension. At each time point, metabolites were extracted from SP:SAMPLEPREP_SUMMARY replicate parasite samples using a modified version of a previously reported SP:SAMPLEPREP_SUMMARY protocol. Briefly, host cell free parasites were collected by centrifugation at SP:SAMPLEPREP_SUMMARY 3000 rpm for 5 minutes in cold and then immediately resuspended in 200 µls of SP:SAMPLEPREP_SUMMARY ice cold 80% acetonitrile (Chem-Impex; JT Bakers) in water and incubated in ice SP:SAMPLEPREP_SUMMARY for 15 minutes with intermittent vortexing. The supernatant was then collected SP:SAMPLEPREP_SUMMARY after centrifuging at 13,000 rpm for 5 minutes and the pellet was further SP:SAMPLEPREP_SUMMARY extracted twice with 100 µls of the same solvent using ultrasound in a SP:SAMPLEPREP_SUMMARY sonicating iced water bath for 15 minutes. All the extracts were pooled (total SP:SAMPLEPREP_SUMMARY 400 µls) and stored in -80°C until further processing for liquid SP:SAMPLEPREP_SUMMARY chromatography coupled mass spectrometry (LC-MS) analysis. SP:EXTRACTION_METHOD Hydrophilic organic extract SP:EXTRACT_STORAGE -80 deg. Cel. until LC-MS analysis is done SP:SAMPLE_RESUSPENSION The acetonitrile:water extracts from parasites were dried under nitrogen flow SP:SAMPLE_RESUSPENSION and resuspended in 200 µls of water:methanol (97:3) containing 10 mM SP:SAMPLE_RESUSPENSION tributylamine and 15 mM acetic acid. SP:SAMPLE_DERIVATIZATION n/a SP:SAMPLE_SPIKING PIPES @ 100ng/ml final concentration #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The acetonitrile:water extracts from parasites were dried under nitrogen flow CH:CHROMATOGRAPHY_SUMMARY and resuspended in 200 µls of water:methanol (97:3) containing 10 mM CH:CHROMATOGRAPHY_SUMMARY tributylamine and 15 mM acetic acid. This solvent was also used as buffer A and CH:CHROMATOGRAPHY_SUMMARY methanol as buffer B for liquid chromatography using a Synergy Hydro-RP column CH:CHROMATOGRAPHY_SUMMARY (Phenomenex) with a bed volume of 100 mm x 2 mm and particle size of 2.5 µ. For CH:CHROMATOGRAPHY_SUMMARY some samples, an alternate method, using a Thermo Accucore C18 column with a bed CH:CHROMATOGRAPHY_SUMMARY volume of 150 mm x 2.1 mm and 2.6 µ particle size. A solvent system composed of CH:CHROMATOGRAPHY_SUMMARY water buffered with 0.1 % formic acid (buffer A) and acetonitrile (buffer B), CH:CHROMATOGRAPHY_SUMMARY was used on a 20 minute gradient run with a flow rate of 200 µl/min as follows- CH:CHROMATOGRAPHY_SUMMARY hold at 10% acetonitrile for 30 seconds and gradually ramp up to 15%, 20%, 50%, CH:CHROMATOGRAPHY_SUMMARY 60% and 90% acetonitrile by 3, 6, 10, 12, 13 minutes, hold at 90% acetonitrile CH:CHROMATOGRAPHY_SUMMARY till 15 minutes, ramp down to 10% acetonitrile by 15.5 minutes and hold till 20 CH:CHROMATOGRAPHY_SUMMARY minutes. LC-MS analysis was done using a Exactive Orbitrap mass spectrometer, CH:CHROMATOGRAPHY_SUMMARY coupled to an Accela U-HPLC (Thermo Fisher Scientific) and HTC PAL autosampler CH:CHROMATOGRAPHY_SUMMARY (CTC Analytics AG). The mass spectrometer was run in negative mode, scanning a CH:CHROMATOGRAPHY_SUMMARY mass-charge ratio (m/z) range of 85-1000. All other parameters used for LC-MS CH:CHROMATOGRAPHY_SUMMARY instrumentation in this study were similar to published protocols. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Accela CH:INSTRUMENT_NAME AG) CH:COLUMN_NAME Synergy Hydro-RP column (Phenomenex) and Accucore C18 column (Thermo) CH:FLOW_GRADIENT A solvent system composed of water buffered with 0.1 % formic acid (buffer A) CH:FLOW_GRADIENT and acetonitrile (buffer B), was used on a 20 minute gradient run with a flow CH:FLOW_GRADIENT rate of 200 µl/min as follows- hold at 10% acetonitrile for 30 seconds and CH:FLOW_GRADIENT gradually ramp up to 15%, 20%, 50%, 60% and 90% acetonitrile by 3, 6, 10, 12, 13 CH:FLOW_GRADIENT minutes, hold at 90% acetonitrile till 15 minutes, ramp down to 10% acetonitrile CH:FLOW_GRADIENT by 15.5 minutes and hold till 20 minutes. CH:FLOW_RATE 200 µl/min CH:COLUMN_TEMPERATURE 22 deg. Cel. CH:SOLVENT_A Water:methanol (97:3) containing 10 mM tributylamine and 15 mM acetic acid for CH:SOLVENT_A Synergy Hydro-RP column. Water buffered with 0.1 % formic acid for accucore C18 CH:SOLVENT_A column. CH:SOLVENT_B Methanol for Synergy Hydro-RP column. Acetonitril for accucore C18 column. CH:COLUMN_PRESSURE 250 to 400 bar CH:INJECTION_TEMPERATURE 22 deg. Cel. CH:INTERNAL_STANDARD PIPES @ 100ng/ml final concentration CH:SAMPLE_INJECTION 10 µl CH:ANALYTICAL_TIME 20 minutes CH:PRECONDITIONING 5-10 minutes CH:TIME_PROGRAM 20 minutes CH:TRANSFERLINE_TEMPERATURE 22 deg. Cel. CH:WASHING_BUFFER Methanol CH:SAMPLE_LOOP_SIZE 10 µl CH:SAMPLE_SYRINGE_SIZE 100 µl #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Molecular Parasitology Laboratory AN:OPERATOR_NAME Dr. Kellen L. Olszewski & Mr. Anurag Shukla AN:DETECTOR_TYPE Orbitrap AN:SOFTWARE_VERSION Xcaliber Ver. 2.2 AN:DATA_FORMAT .RAW #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_TEMPERATURE 320 deg. Cel. MS:CAPILLARY_VOLTAGE -3.0 kv MS:ION_SOURCE_TEMPERATURE 250 deg. Cel. MS:IONIZATION Negative MS:MASS_ACCURACY <1 ppm MS:REAGENT_GAS Nitrogen MS:DATAFORMAT .RAW MS:RESOLUTION_SETTING 70,000 MS:SCAN_RANGE_MOVERZ 100 to 1000 Da MS:SCANNING_RANGE Full MS MS:MS_RESULTS_FILE ST000817_AN001295_Results.xlsx UNITS:NA #END