{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001275","ANALYSIS_ID":"AN002116","VERSION":"1","CREATED_ON":"November 18, 2019, 2:14 pm"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics-based profiling of the mode of action of Pathogen Box compounds in Trypanosoma brucei","PROJECT_SUMMARY":"The mode of action of anti-Trypanosomal compounds from the Pathogen Box was investigated using an unbiased metabolomics approach. Trypanosoma brucei parasites were incubated for five hours with the test compounds at 0.5 µM concentration. Analysis of cell pellets allowed reproducible detection of diverse metabolites from a range of metabolic pathways.","INSTITUTE":"Monash University","LAST_NAME":"Creek","FIRST_NAME":"Darren","ADDRESS":"Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia","EMAIL":"darren.creek@monash.edu","PHONE":"+61 (0) 3 9903 9249"},

"STUDY":{"STUDY_TITLE":"Metabolomics-based profiling of the mode of action of Pathogen Box compounds in Trypanosoma brucei (part-II)","STUDY_SUMMARY":"The mode of action of anti-Trypanosomal compounds from the Pathogen Box was investigated using an unbiased metabolomics approach. Trypanosoma brucei parasites were incubated for five hours with the test compounds at 0.5 µM concentration. Analysis of cell pellets allowed reproducible detection of diverse metabolites from a range of metabolic pathways.","INSTITUTE":"Monash University","LAST_NAME":"Creek","FIRST_NAME":"Darren","ADDRESS":"Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia","EMAIL":"darren.creek@monash.edu","PHONE":"+61 (0) 3 9903 9249"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Trypanosoma brucei brucei","TAXONOMY_ID":"5702","GENOTYPE_STRAIN":"427"},
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"Factors":{"Drug treatment":"MMV688797"}
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{
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"Subject ID":"MMV690028",
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],
"COLLECTION":{"COLLECTION_SUMMARY":"Trypanosoma brucei brucei (T.b.b) bloodstream forms (strain 427) were maintained in vitro in 5-10 ml cultures at 37 °C and 5% CO2 in Creeks minimal media supplemented with 10% HMI-9. The cultures were passaged every 2-3 days and cells were grown to a maximum density of 2x106cells/ml.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"For drug-induced metabolic perturbation experiments, cells were sub-cultured at 10e6cells/ml in 20 ml volume with drugs added at 0.5 µM concentration and incubated for a further 5 hours until cell density reached ~2x10e6cells/ml. Cells were then used for metabolite quenching and extraction."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Metabolism was rapidly quenched by rapidly cooling cultures to 4°C in a dry ice and ethanol bath. Cultures were then centrifuged for 10 minutes at 1250g at 4°C. The supernatant was discarded and cells were washed with 1 ml of cold PBS by centrifugation for 1 minute at 2100g at 4°C. The supernatant was removed and cells were extracted with 100 µl extraction solvent containing chloroform: methanol: water (1:3:1 v/v) followed by vortexing at 4 °C for 1 hour. The resulting suspension was centrifuged for 10 minutes at 2100g at 4°C and the supernatant was transferred to glass vials and stored at -80 °C until analysis by liquid chromatography and high-resolution mass spectrometry."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Metabolite extracts were analysed using reversed phase liquid chromatography (LC) and high resolution mass spectrometry on an Orbitrap system.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000 RS","COLUMN_NAME":"Ascentis Express C8, 2.7 µm HPLC Column (2.7 µm particle size, L × I.D. 10 cm × 2.1 mm)","FLOW_GRADIENT":"linear gradient -time, %B as follows: 0min- 0%, 1.5min- 20%, 7min- 28%, 8min- 35%, 24min- 65%, 25min- 100%.","FLOW_RATE":"200 μL/min","COLUMN_TEMPERATURE":"40°C","SOLVENT_A":"40% (v/v) isopropanol, 2 mM formic acid, 8 mM ammonium formate","SOLVENT_B":"98% (v/v) isopropanol, 2 mM formic acid, 8 mM ammonium formate","SAMPLE_INJECTION":"10 μL"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"The raw LC-MS data was processed using IDEOM.","CAPILLARY_TEMPERATURE":"300°C","SPRAY_VOLTAGE":"3.5kV","RESOLUTION_SETTING":"70000","SCAN_RANGE_MOVERZ":"140-1300 m/z"},

"MS_METABOLITE_DATA":{
"Units":"Peak height",

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A2","Mass":"334.2144485","RT":"8.658887902","confidence":"8"},{"Metabolite":"SM(d32:0)","Mass":"676.5514687","RT":"13.12694099","confidence":"7"},{"Metabolite":"SM(d36:0)","Mass":"732.6153166","RT":"15.79994687","confidence":"5"},{"Metabolite":"SM(d36:1)","Mass":"730.5991874","RT":"15.24047747","confidence":"5"},{"Metabolite":"Choline phosphate","Mass":"183.0659131","RT":"13.8537159","confidence":"6"},{"Metabolite":"[PR] Abietic acid","Mass":"302.224521","RT":"7.864155565","confidence":"8"},{"Metabolite":"[PR] Taxa-4(20),11(12)-dien-5alpha-ol","Mass":"288.2449224","RT":"7.348135197","confidence":"8"},{"Metabolite":"Dehydroabietic acid","Mass":"300.2089079","RT":"7.475310538","confidence":"7"},{"Metabolite":"FA oxo(12:1)","Mass":"212.1414036","RT":"4.994239183","confidence":"6"},{"Metabolite":"[FA (18:1)] 9Z-octadecenoic acid","Mass":"282.2560171","RT":"10.72632263","confidence":"8"},{"Metabolite":"[FA (18:3)] 6Z,9Z,12Z-octadecatrienoic acid","Mass":"278.2245244","RT":"5.81580907","confidence":"8"},{"Metabolite":"[FA (20:5)] 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid","Mass":"302.2248597","RT":"9.150194845","confidence":"8"},{"Metabolite":"[PR] (-)-Carvone","Mass":"150.1044096","RT":"9.791155435","confidence":"6"},{"Metabolite":"[PR] Tretinoin/All-Trans Retinoic Acid","Mass":"300.2088433","RT":"6.396042833","confidence":"8"},{"Metabolite":"[SP hydrox] 4-hydroxysphinganine","Mass":"317.2930489","RT":"8.963171927","confidence":"6"},{"Metabolite":"[SP] 3-dehydrosphinganine","Mass":"299.2824612","RT":"9.655664187","confidence":"6"},{"Metabolite":"[SP] Sphing-4-enine","Mass":"299.282414","RT":"10.47604139","confidence":"8"},{"Metabolite":"[SP] Sphinganine-1-phosphate","Mass":"381.2641424","RT":"6.141210705","confidence":"8"},{"Metabolite":"[ST (2:0)] (5Z,7E)-(3S)-9,10-seco-5,7,10(19)-cholestatriene-3,25-diol","Mass":"400.3341605","RT":"9.597431428","confidence":"6"},{"Metabolite":"[ST (3:0)] cholest-5Z,7Z,24-trien-3beta-ol","Mass":"382.3236449","RT":"12.82485318","confidence":"6"},{"Metabolite":"[ST hydrox] 3alpha-hydroxy-5alpha-androstan-17-one","Mass":"290.2246076","RT":"5.864384938","confidence":"7"},{"Metabolite":"[ST hydrox] 3alpha-hydroxy-5beta-androstan-17-one","Mass":"290.2245952","RT":"7.419283192","confidence":"7"},{"Metabolite":"[ST hydrox] 3beta-hydroxypregn-5-ene-20-one","Mass":"316.24024","RT":"8.22340319","confidence":"8"},{"Metabolite":"[ST] cholest-7-en-3beta-ol","Mass":"386.3550279","RT":"10.37238566","confidence":"6"},{"Metabolite":"Cholesta-5,7-dien-3beta-ol","Mass":"384.3392207","RT":"13.02760185","confidence":"6"},{"Metabolite":"Estradiol-17beta","Mass":"272.1778502","RT":"5.144771947","confidence":"8"},{"Metabolite":"FA (20:0)","Mass":"312.3026822","RT":"13.26767262","confidence":"8"},{"Metabolite":"FA (20:4)","Mass":"304.2404424","RT":"9.975024137","confidence":"8"},{"Metabolite":"Hexadecanoic acid","Mass":"256.2402926","RT":"10.19231362","confidence":"8"},{"Metabolite":"Linoleate","Mass":"280.2403386","RT":"9.861070135","confidence":"8"},{"Metabolite":"Octadecanoic acid","Mass":"284.2714941","RT":"11.70646739","confidence":"8"},{"Metabolite":"O-Palmitoyl-R-carnitine","Mass":"399.3348278","RT":"6.133687685","confidence":"8"},{"Metabolite":"Palmitoyl-CoA","Mass":"1005.344569","RT":"5.035983337","confidence":"6"},{"Metabolite":"Sphinganine","Mass":"301.2979961","RT":"11.70397722","confidence":"6"},{"Metabolite":"Tetradecanoic acid","Mass":"228.2089996","RT":"12.27745293","confidence":"8"},{"Metabolite":"[FA (20:2)] hydroperoxy-eicosadienoic acid","Mass":"340.2614208","RT":"10.78046597","confidence":"7"},{"Metabolite":"[FA hydroxy(20:4)] eicosatetraenoyl amine","Mass":"319.2511869","RT":"9.15768512","confidence":"7"},{"Metabolite":"[FA hydroxy(20:4)] N-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoyl amine","Mass":"319.2511411","RT":"10.53269067","confidence":"7"},{"Metabolite":"[FA hydroxy(3:2/20:4)] N-(3-hydroxy-propyl)-5Z,8Z,11Z,14Z-eicosatetraenoyl amine","Mass":"361.2980418","RT":"12.37900259","confidence":"7"},{"Metabolite":"[FA methoxy,hydroxy(18:2)] octadecadienoic acid","Mass":"326.2456613","RT":"8.588391948","confidence":"7"},{"Metabolite":"[PC(21:4)] 1-(6Z,9Z,12Z,15Z-heneicosatetraenoyl)-sn-glycero-3-phosphocholine","Mass":"557.3482855","RT":"7.184598817","confidence":"5"},{"Metabolite":"[PC(22:6)]","Mass":"567.3324807","RT":"6.292328748","confidence":"7"},{"Metabolite":"[PE(O-18:2)] 1-octadecyl-sn-glycero-3-phosphoethanolamine","Mass":"467.3382659","RT":"8.711036145","confidence":"7"},{"Metabolite":"[PG hydrox] 1-(3-hydroxyphytanyl)-2-phytanyl-sn-glycero-3-phospho-(1'-sn-glycerol)","Mass":"822.6721704","RT":"22.84345648","confidence":"5"},{"Metabolite":"[PI(18:0)] 1-octadecanoyl-sn-glycero-3-phospho-(1'-myo-inositol)","Mass":"600.3274869","RT":"6.610771965","confidence":"7"},{"Metabolite":"[PR tetramethyl(4:0/16:1)] 3R,7R,11R,15-tetramethyl-2E-hexadecen-1-ol phosphate","Mass":"376.2746064","RT":"10.31127205","confidence":"7"},{"Metabolite":"[PR] bacteriohopane-,32,33,34-triol-35-cyclitolguanine","Mass":"749.556323","RT":"13.5102344","confidence":"5"},{"Metabolite":"[SP (16:0)] N-(hexadecanoyl)-sphinganine-1-phosphocholine","Mass":"704.5824581","RT":"14.38004059","confidence":"7"},{"Metabolite":"[SP (26:0)] N-(17Z-hexacosenoyl)-sphing-4-enine-1-phosphocholine","Mass":"840.7092172","RT":"19.9854473","confidence":"5"},{"Metabolite":"[SP (3:0)] sphinga-4E,8E,10E-trienine","Mass":"295.2512245","RT":"9.014122678","confidence":"7"},{"Metabolite":"[SP amino,dimethyl(18:0)] 2-amino-14,16-dimethyloctadecan-3-ol","Mass":"313.3343374","RT":"12.7865344","confidence":"7"},{"Metabolite":"[SP amino,hydroxy(4:0/20:0)] 2S-amino-3R,4R,5S,14R-tetrahydroxyeicos-6E-enoic acid","Mass":"389.2778012","RT":"6.17901253","confidence":"7"},{"Metabolite":"[SP amino,trihydroxy(20:0)] 2S-amino-5S-acetoxy-3R,4R,14R-trihydroxyeicos-6E-enoic acid","Mass":"431.2881991","RT":"8.130658733","confidence":"7"},{"Metabolite":"[SP diamino,trihydroxy(28:0/28:0)] diamino-trihydroxyoctacosan-11-one","Mass":"486.4399929","RT":"11.0308744","confidence":"5"},{"Metabolite":"[SP dimethyl,amino(18:0/2:0)] 2S-dimethylaminooctadecane-1,3R-diol","Mass":"329.3294733","RT":"5.864241108","confidence":"7"},{"Metabolite":"[SP methyl(10:0/8:0/8:0)] (9aS)-6S-(deca-1,3E-dienyl)-4S-methyloctahydro-1H-quinolizin-3R-ol","Mass":"305.2716165","RT":"9.470439228","confidence":"7"},{"Metabolite":"[SP methyl(2:0)] 9-methyl-sphinga-4E,8E-dienine","Mass":"311.282234","RT":"11.63168405","confidence":"7"},{"Metabolite":"[SP] 1-deoxy-sphinganine","Mass":"285.3031224","RT":"6.520353382","confidence":"7"},{"Metabolite":"[ST (3:0)] (5Z,7E)-(3S)-3-azido-9,10-seco-5,7,10(19)-cholestatrien-25-ol","Mass":"426.3479004","RT":"10.31863299","confidence":"5"},{"Metabolite":"[ST hydroxy(6:2/3:0)] (5Z,7E)-(1S,2R,3R)-2-(6-hydroxyhexyl)-9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol","Mass":"516.4183159","RT":"17.79202727","confidence":"7"},{"Metabolite":"[ST] (5Z,7E)-9,10-seco-5,7,10(19)-cholestatriene","Mass":"368.3439099","RT":"12.94947683","confidence":"7"},{"Metabolite":"1-(14-methyl-pentadecanoyl)-2-(8-[3]-ladderane-octanyl)-sn-glycerol","Mass":"602.5263936","RT":"14.04410823","confidence":"5"},{"Metabolite":"1-(9E-decenyl)-sn-glycero-3-phosphocholine","Mass":"395.2437179","RT":"8.90626831","confidence":"5"},{"Metabolite":"1,9S,11R,15S-tetrahydroxy-13E-prostaene","Mass":"342.2770911","RT":"11.07356522","confidence":"7"},{"Metabolite":"3-O-(6'-O-octadecanoyl-beta-D-glucopyranosyl)-campest-5-en-3beta-ol","Mass":"828.6843106","RT":"21.76730132","confidence":"5"},{"Metabolite":"5S-HETE di-endoperoxide","Mass":"402.2254141","RT":"6.700406585","confidence":"5"},{"Metabolite":"6Z,9Z-Eicosadien-11-ol","Mass":"294.2923712","RT":"9.017586415","confidence":"7"},{"Metabolite":"Cer(d34:2))","Mass":"535.4967718","RT":"15.90081244","confidence":"5"},{"Metabolite":"Cer(d40:2)","Mass":"619.5905351","RT":"20.30507753","confidence":"5"},{"Metabolite":"cholest-5,24-dien-3beta-ol 3-O-beta-D-glucopyranoside","Mass":"546.3922889","RT":"17.87178173","confidence":"7"},{"Metabolite":"Dehydrophytosphingosine","Mass":"315.2772358","RT":"6.168309958","confidence":"7"},{"Metabolite":"DG(34:1)","Mass":"594.5224036","RT":"9.84789228","confidence":"5"},{"Metabolite":"FA (22:0)","Mass":"314.3185199","RT":"8.854444913","confidence":"7"},{"Metabolite":"FA hydroxy(18:1)]","Mass":"314.2455824","RT":"9.713363135","confidence":"5"},{"Metabolite":"FA hydroxy(22:0)","Mass":"356.3290602","RT":"10.44918724","confidence":"7"},{"Metabolite":"Linoleamide","Mass":"279.2562729","RT":"8.218585392","confidence":"7"},{"Metabolite":"LysoPC(18:1)","Mass":"521.3486164","RT":"6.99001561","confidence":"5"},{"Metabolite":"LysoPC(18:2)","Mass":"505.3537569","RT":"7.679463835","confidence":"7"},{"Metabolite":"LysoPC(O-16:1)","Mass":"479.3378326","RT":"7.18853159","confidence":"7"},{"Metabolite":"LysoPE(18:1)","Mass":"465.3221328","RT":"8.910443488","confidence":"7"},{"Metabolite":"Margaroyl-EA","Mass":"313.2979546","RT":"12.61243011","confidence":"7"},{"Metabolite":"N-(11Z,14Z,17Z-eicosatrienoyl)-ethanolamine","Mass":"349.2978862","RT":"12.412288","confidence":"7"},{"Metabolite":"N-(11Z,14Z-eicosadienoyl)-ethanolamine","Mass":"351.3137158","RT":"7.70634033","confidence":"7"},{"Metabolite":"N-(11Z-eicosaenoyl)-ethanolamine","Mass":"353.329392","RT":"8.75290207","confidence":"7"},{"Metabolite":"N-(11Z-eicosenoyl)-tetradecasphing-4-enine-1-phosphoethanolamine","Mass":"658.505248","RT":"13.32105272","confidence":"5"},{"Metabolite":"N-(2-hydroxy-13Z-docosenoyl)-4E,6E-tetradecasphingadienine-1-phosphoethanolamine","Mass":"700.5159853","RT":"13.49644186","confidence":"7"},{"Metabolite":"N-(2-hydroxy-docosanoyl)-pentadecasphing-4-enine-1-phosphoethanolamine","Mass":"718.5625661","RT":"12.97526783","confidence":"7"},{"Metabolite":"N-(2-hydroxy-docosanoyl)-tetradecasphing-4-enine-1-phosphoethanolamine","Mass":"704.5469406","RT":"11.57693791","confidence":"7"},{"Metabolite":"N-(2-hydroxy-eicosanoyl)-pentadecasphing-4-enine-1-phosphoethanolamine","Mass":"690.5309438","RT":"11.83300055","confidence":"7"},{"Metabolite":"N-(2-hydroxy-eicosanoyl)-tetradecasphing-4-enine-1-phosphoethanolamine","Mass":"676.5151736","RT":"12.7866909","confidence":"5"},{"Metabolite":"N-(2-hydroxy-hexacosanoyl)-tetradecasphing-4-enine","Mass":"637.6011366","RT":"21.4004819","confidence":"5"},{"Metabolite":"N-(2-hydroxy-tetracosanoyl)-4E,6E-hexadecasphingadienine","Mass":"635.58568","RT":"21.38502637","confidence":"5"},{"Metabolite":"N-(2-hydroxy-tetracosanoyl)-tetradecasphing-4-enine-1-phosphoethanolamine","Mass":"732.5770533","RT":"13.21526832","confidence":"5"},{"Metabolite":"N-(2R-methyl-3-hydroxy-ethyl)-16,16-dimethyl-5Z,8Z,11Z,14Z-docosatetraenoyl 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