#METABOLOMICS WORKBENCH michaelsa93_20170106_122829 DATATRACK_ID:815 STUDY_ID:ST000548 ANALYSIS_ID:AN000836 PROJECT_ID:PR000402 VERSION 1 CREATED_ON February 8, 2017, 1:23 pm #PROJECT PR:PROJECT_TITLE Reproducibility study: The common feature of leukemia-associated IDH1 and IDH2 PR:PROJECT_TITLE mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to PR:PROJECT_TITLE 2-hydroxyglutarate PR:PROJECT_SUMMARY The Reproducibility Project: Cancer Biology seeks to address growing concerns PR:PROJECT_SUMMARY about reproducibility in scientific research by conducting replications of PR:PROJECT_SUMMARY selected experiments from a number of high-profile papers in the field of cancer PR:PROJECT_SUMMARY biology. The papers, which were published between 2010 and 2012, were selected PR:PROJECT_SUMMARY on the basis of citations and Altmetric scores (Errington et al., 2014). This PR:PROJECT_SUMMARY Registered Report describes the proposed replication plan of key experiments PR:PROJECT_SUMMARY from “The common feature of leukemia-associated IDH1 and IDH2 mutations is a PR:PROJECT_SUMMARY neomorphic enzyme activity converting alpha-ketoglutarate to PR:PROJECT_SUMMARY 2-hydroxyglutarate” by Ward and colleagues, published in Cancer Cell in 2010 PR:PROJECT_SUMMARY (Ward et al., 2010). The experiments that will be replicated are those reported PR:PROJECT_SUMMARY in Figures 2, 3 and 5. Ward and colleagues demonstrate the mutations in PR:PROJECT_SUMMARY isocitrate dehydrogenase 2 (IDH2), commonly found in acute myeloid leukemia PR:PROJECT_SUMMARY (AML), abrogate the enzyme’s wild-type activity and confer to the mutant PR:PROJECT_SUMMARY neomorphic activity that produces the oncometabolite 2-hydroxyglutarate (2-HG) PR:PROJECT_SUMMARY (Figures 2 and 3). They then show that elevated levels of 2-HG are correlated PR:PROJECT_SUMMARY with mutations in IDH1 and IDH2in AML patient samples (Figure 5). The PR:PROJECT_SUMMARY Reproducibility Project: Cancer Biology is a collaboration between the Center PR:PROJECT_SUMMARY for Open Science and Science Exchange and the results of the replications PR:PROJECT_SUMMARY will be published by eLife. PR:INSTITUTE University of California, Davis PR:DEPARTMENT Genome and Biolmedical Sciences Facility PR:LABORATORY WCMC Metabolomics Core PR:LAST_NAME Fiehn PR:FIRST_NAME Oliver PR:ADDRESS Health Sciences Drive, Davis, California, 95616, USA PR:EMAIL ofiehn@ucdavis.edu PR:PHONE (530) 754-8258 PR:FUNDING_SOURCE NIH U24DK097154 #STUDY ST:STUDY_TITLE Replication study: The common feature of leukemia-associated IDH1 and IDH2 ST:STUDY_TITLE mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to ST:STUDY_TITLE 2-hydroxyglutarate ST:STUDY_SUMMARY The Reproducibility Project: Cancer Biology seeks to address growing concerns ST:STUDY_SUMMARY about reproducibility in scientific research by conducting replications of ST:STUDY_SUMMARY selected experiments from a number of high-profile papers in the field of cancer ST:STUDY_SUMMARY biology. The papers, which were published between 2010 and 2012, were selected ST:STUDY_SUMMARY on the basis of citations and Altmetric scores (Errington et al., 2014). This ST:STUDY_SUMMARY Registered Report describes the proposed replication plan of key experiments ST:STUDY_SUMMARY from “The common feature of leukemia-associated IDH1 and IDH2 mutations is a ST:STUDY_SUMMARY neomorphic enzyme activity converting alpha-ketoglutarate to ST:STUDY_SUMMARY 2-hydroxyglutarate” by Ward and colleagues, published in Cancer Cell in 2010 ST:STUDY_SUMMARY (Ward et al., 2010). The experiments that will be replicated are those reported ST:STUDY_SUMMARY in Figures 2, 3 and 5. Ward and colleagues demonstrate the mutations in ST:STUDY_SUMMARY isocitrate dehydrogenase 2 (IDH2), commonly found in acute myeloid leukemia ST:STUDY_SUMMARY (AML), abrogate the enzyme’s wild-type activity and confer to the mutant ST:STUDY_SUMMARY neomorphic activity that produces the oncometabolite 2-hydroxyglutarate (2-HG) ST:STUDY_SUMMARY (Figures 2 and 3). They then show that elevated levels of 2-HG are correlated ST:STUDY_SUMMARY with mutations in IDH1 and IDH2in AML patient samples (Figure 5). The ST:STUDY_SUMMARY Reproducibility Project: Cancer Biology is a collaboration between the Center ST:STUDY_SUMMARY for Open Science and Science Exchange and the results of the replications ST:STUDY_SUMMARY will be published by eLife. ST:INSTITUTE University of California, Davis ST:DEPARTMENT Genome and Biomedical Sciences Facility ST:LABORATORY WCMC Metabolomics Core ST:LAST_NAME Fiehn ST:FIRST_NAME Oliver ST:ADDRESS Health Sciences Drive, Davis, California, 95616, USA ST:EMAIL ofiehn@ucdavis.edu ST:PHONE (530) 754-8258 #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Inj08_STD6_liner2.D QC001 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj09_CS29_V09_24.D V09 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj10_CS19_MUT09_24.D MUT09 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj11_CS17_MUT07_24.D MUT07 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj12_CS16_MUT06_24.D MUT06 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj13_CS02_WT02_24.D WT02 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj14_CS07_WT07_24.D WT07 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj15_CS05_WT05_24.D WT05 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj16_CS14_MUT04_24.D MUT04 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj17_CS27_V07_24.D V07 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj18_CS18_MUT08_24.D MUT08 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj19_STD6_liner2.D QC002 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj20_CS04_WT04_24.D WT04 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj21_CS06_WT06_24.D WT06 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj22_CS22_V02_24.D V02 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj23_CS03_WT03_24.D WT03 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj24_CS20_MUT10_24.D MUT10 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj25_CS10_WT10_24.D WT10 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj26_CS01_WT01_24.D WT01 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj27_CS15_MUT05_24.D MUT05 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj28_CS08_WT08_24.D WT08 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj29_CS25_V05_24.D V05 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj30_STD6_liner2.D QC003 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj31_CS13_MUT03_24.D MUT03 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj32_CS09_WT09_24.D WT09 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj33_CS30_V10_24.D V10 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj34_CS26_V06_24.D V06 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj35_CS24_V04_24.D V04 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj36_CS21_V01_24.D V01 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj37_CS12_MUT02_24.D MUT02 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj38_CS23_V03_24.D V03 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj39_CS11_MUT01_24.D MUT01 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj40_CS28_V08_24.D V08 Time Point:CS-24h | Sample Source:293T HEK cell line Sample Type=Vector SUBJECT_SAMPLE_FACTORS Inj41_STD6_liner2.D QC004 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj43_CS33_MB_01.D Blank001 Time Point:Blank | Sample Source:- Sample Type=MTBSTFA/ACN (1:1,v/v) SUBJECT_SAMPLE_FACTORS Inj45_STD6_liner2.D QC005 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj46_HR_11_IDH1.D HR_11_IDH1 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH1 Mutant (R132C) SUBJECT_SAMPLE_FACTORS Inj47_HR_09_IDH1.D HR_09_IDH1 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH1 Mutant (R132C) SUBJECT_SAMPLE_FACTORS Inj48_HR_02_WT.D HR_02_WT Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH Wild Type SUBJECT_SAMPLE_FACTORS Inj49_HR_05_IDH2.D HR_05_IDH2 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj50_HR_07_IDH2.D HR_07_IDH2 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj51_HR_10_IDH1.D HR_10_IDH1 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH1 Mutant (R132C) SUBJECT_SAMPLE_FACTORS Inj52_STD6_liner2.D QC06 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj53_HR_08_IDH2.D HR_08_IDH2 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj54_HR_12_IDH1.D HR_12_IDH1 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH1 Mutant (R132C) SUBJECT_SAMPLE_FACTORS Inj55_HR_03_WT.D HR_03_WT Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH Wild Type SUBJECT_SAMPLE_FACTORS Inj56_HR_06_IDH2.D HR_06_IDH2 Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj57_HR_04_WT.D HR_04_WT Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH Wild Type SUBJECT_SAMPLE_FACTORS Inj58_HR_01_WT.D HR_01_WT Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Sample Type=IDH Wild Type SUBJECT_SAMPLE_FACTORS Inj59_HR_MB.D Blank002 Time Point:Blank | Sample Source:- Sample Type=MTBSTFA/ACN (1:1,v/v) SUBJECT_SAMPLE_FACTORS Inj60_STD6_liner2.D QC007 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj08_STD6_liner3.D QC008 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj09_CS49_MUT09.D CS49_MUT09_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj10_CS43_MUT03.D CS43_MUT03_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj11_CS56_V06.D CS56_V06_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj12_CS53_V03.D CS53_V03_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj13_CS37_WT07.D CS37_WT07_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj14_CS47_MUT07.D CS47_MUT07_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj15_CS58_V08.D CS58_V08_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj16_CS51_V01.D CS51_V01_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj17_CS33_WT03.D CS33_WT03_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj18_CS44_MUT04.D CS44_MUT04_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj19_STD6_liner3.D QC009 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj20_CS41_MUT01.D CS41_MUT01_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj21_CS39_WT09.D CS39_WT09_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj22_CS52_V02.D CS52_V02_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj23_CS48_MUT08.D CS48_MUT08_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj24_CS42_MUT02.D CS42_MUT02_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj25_CS57_V07.D CS57_V07_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj26_CS45_MUT05.D CS45_MUT05_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj27_CS34_WT04.D CS34_WT04_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj28_CS36_WT06.D CS36_WT06_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj29_CS50_MUT10.D CS50_MUT10_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj30_STD6_liner3.D QC010 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj31_CS40_WT10.D CS40_WT10_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj32_CS38_WT08.D CS38_WT08_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj33_CS55_V05.D CS55_V05_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj34_CS59_V09.D CS59_V09_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj35_CS54_V04.D CS54_V04_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj36_CS35_WT05.D CS35_WT05_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj37_CS46_MUT06.D CS46_MUT06_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Mutant (R172K) SUBJECT_SAMPLE_FACTORS Inj38_CS32_WT02.D CS32_WT02_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj39_CS60_V10.D CS60_V10_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=Empty Vector Control SUBJECT_SAMPLE_FACTORS Inj40_CS31_WT01.D CS31_WT01_48 Time Point:CS-48h | Sample Source:293T HEK cell line Sample Type=IDH2 Wild Type SUBJECT_SAMPLE_FACTORS Inj41_STD6_liner3.D QC011 Time Point:QC | Sample Source:- Sample Type=Quality Control SUBJECT_SAMPLE_FACTORS Inj44_MB_liner3.D Blank003 Time Point:Blank | Sample Source:- Sample Type=MTBSTFA/ACN (1:1,v/v) #COLLECTION CO:COLLECTION_SUMMARY 293T Cells were harvested 24 and 48 hours after transfection by removal of media CO:COLLECTION_SUMMARY and rapid quenching with 1.5 mL per 10 cm plate of -80°C methanol. Cells were CO:COLLECTION_SUMMARY incubated at -80°C for 15 minutesmin, then scraped off the dish and centrifuged CO:COLLECTION_SUMMARY for 5 min at 2,000 x g at 4°C to pellet cellular debris. Pellet was CO:COLLECTION_SUMMARY re-extracted by addition of 500 µL of -80°C 80% methanol in water, vortexed, CO:COLLECTION_SUMMARY then incubated at 4°C for 15 minutes and centrifuged for 5 minutes at 2,000 x g CO:COLLECTION_SUMMARY at 4°C. Peripheral blood mono-nuclear cells were centrifuged at 2,000 x g, CO:COLLECTION_SUMMARY freeze media removed and extracted using same procedure as 293T cells. CO:COLLECTION_PROTOCOL_FILENAME The common feature of IDH1 and IDH2 mutations.pdf CO:SAMPLE_TYPE Cells CO:COLLECTION_LOCATION UC Davis Genome and Biomedical Sciences Facility #TREATMENT TR:TREATMENT_SUMMARY 239T cells (ATCC CRL-3216) were grown in DMEM (Invitrogen) with 10% FBS TR:TREATMENT_SUMMARY (Hyclone) at -37°C in 10% CO2 and transfected with pcDNA3.1, pcDNA3.1-IDH2WT, TR:TREATMENT_SUMMARY pcDNA3.1-IDH2R172K (Invitrogen and Origene) with Lipofectamine 2000 (Invitrogen) TR:TREATMENT_SUMMARY according to manufacturer instructions. 6x10^5 cells were seeded in 6 well TR:TREATMENT_SUMMARY plates for protocol 1 and 3.5x10^6 cells were seeded in 10 cm plates for TR:TREATMENT_SUMMARY protocol 2. Identity of all vectors was confirmed by sequencing and vector TR:TREATMENT_SUMMARY integrity with agarose gel electrophoresis. TR:TREATMENT_PROTOCOL_FILENAME The common feature of IDH1 and IDH2 mutations.pdf TR:CELL_ENVIR_COND 239T cells (ATCC CRL-3216) were grown in DMEM (Invitrogen) with 10% FBS TR:CELL_ENVIR_COND (Hyclone) at 37°C in 10% CO2 and transfected with pcDNA3.1, pcDNA3.1-IDH2WT, TR:CELL_ENVIR_COND pcDNA3.1-IDH2R172K (Invitrogen and Origene) with Lipofectamine 2000 (Invitrogen) TR:CELL_ENVIR_COND according to manufacturer instructions. TR:CELL_HARVESTING 24-48 hours after transfection #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Supernatants were combined and evaporated to dryness. Samples were then SP:SAMPLEPREP_SUMMARY re-suspended in 200 µL LC-MS-grade water. A 2 mL AG-1 X8 100-200 anion exchange SP:SAMPLEPREP_SUMMARY resin (Bio-Rad) was washed with 5 column volumes (10 mL) of 3 N HCl followed by SP:SAMPLEPREP_SUMMARY transfer of re-suspended extracts to resin column. Metabolites were eluted using SP:SAMPLEPREP_SUMMARY 10 mL 3 N HCl. Samples were evaporated to dryness and re-suspended in 100 µL SP:SAMPLEPREP_SUMMARY MTBSTFA/ACN (1:1, v/v) and shaken at 60°C for 1 hour. Derivatized extracts were SP:SAMPLEPREP_SUMMARY further diluted (1:4) with a MTBSTFA/ACN (1:1, v/v) mixture and transferred to SP:SAMPLEPREP_SUMMARY glass vials with micro-inserts and capped immediately. SP:SAMPLEPREP_PROTOCOL_FILENAME The common feature of IDH1 and IDH2 mutations.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent HP5-MS (30m × 0.25mm, 0.25 um) CH:FLOW_RATE 1mL/min CH:COLUMN_TEMPERATURE 250°C CH:METHODS_FILENAME Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf CH:SAMPLE_INJECTION 0.2µl CH:OVEN_TEMPERATURE 100°C (3 min), 4°C/min to 230°C (hold 4 min), 30°C/min to 300°C (hold 5 CH:OVEN_TEMPERATURE min) CH:TRANSFERLINE_TEMPERATURE 230°C CH:RANDOMIZATION_ORDER Excel generated #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME WCMC Metabolomics Center AN:DETECTOR_TYPE TOF AN:SOFTWARE_VERSION Chromatof AN:DATA_FORMAT .d #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:ION_SOURCE_TEMPERATURE 230°C MS:IONIZATION_ENERGY 70eV MS:MASS_ACCURACY Nominal MS:SCAN_RANGE_MOVERZ 50-600 Da MS:SCANNING_CYCLE 2.71 scans/sec MS:SCANNING_RANGE 50-600 Da #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Counts MS_METABOLITE_DATA_START Samples V09 MUT09 MUT07 MUT06 WT02 WT07 WT05 MUT04 V07 MUT08 QC002 WT04 WT06 V02 WT03 MUT10 WT10 WT01 MUT05 WT08 V05 QC003 MUT03 WT09 V10 V06 V04 V01 MUT02 V03 MUT01 V08 QC004 Blank001 QC005 HR_11_IDH1 HR_09_IDH1 HR_02_WT HR_05_IDH2 HR_07_IDH2 HR_10_IDH1 QC06 HR_08_IDH2 HR_12_IDH1 HR_03_WT HR_06_IDH2 HR_04_WT HR_01_WT Blank002 QC007 QC008 CS49_MUT09_48 CS43_MUT03_48 CS56_V06_48 CS53_V03_48 CS37_WT07_48 CS47_MUT07_48 CS58_V08_48 CS51_V01_48 CS33_WT03_48 CS44_MUT04_48 QC009 CS41_MUT01_48 CS39_WT09_48 CS52_V02_48 CS48_MUT08_48 CS42_MUT02_48 CS57_V07_48 CS45_MUT05_48 CS34_WT04_48 CS36_WT06_48 CS50_MUT10_48 QC010 CS40_WT10_48 CS38_WT08_48 CS55_V05_48 CS59_V09_48 CS54_V04_48 CS35_WT05_48 CS46_MUT06_48 CS32_WT02_48 CS60_V10_48 CS31_WT01_48 QC011 Blank003 Factors Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:QC | Sample Source:- Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:QC | Sample Source:- Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:CS-24h | Sample Source:293T HEK cell line Time Point:QC | Sample Source:- Time Point:Blank | Sample Source:- Time Point:QC | Sample Source:- Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:QC | Sample Source:- Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:HR | Sample Source:Peripheral blood mono-nuclear cells Time Point:Blank | Sample Source:- Time Point:QC | Sample Source:- Time Point:QC | Sample Source:- Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:QC | Sample Source:- Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:QC | Sample Source:- Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:CS-48h | Sample Source:293T HEK cell line Time Point:QC | Sample Source:- Time Point:Blank | Sample Source:- Asp TIC Results 62067.0586 68997.33993 75371.38813 70801.55076 57925.81691 54193.40238 70498.59874 75943.91992 64545.09647 85047.2726 447866.2448 48154.70257 57527.10983 195115.1489 109874.8587 24718.9052 42896.08221 65546.02794 66255.50981 52567.21863 59880.8502 517862.6181 184595.3644 68731.63646 69226.80163 69381.23022 50766.08415 66367.8631 89576.99165 75550.29143 82785.95803 54247.98495 429798.3593 5306.82436 508958.2916 609124.4604 199059.426 498470.7185 114465.8525 294486.5884 148462.0829 830592.7041 1251014.126 30251.68959 135569.8079 493269.6016 3450604.433 207144.2845 10665.68855 788290.8294 620218.6602 22127.07494 55695.28688 27501.0992 48423.55338 21037.22373 22634.30231 24947.40435 24803.51163 33477.10421 15504.29583 586575.8508 23392.70818 22287.02709 22427.61428 22521.52089 31661.43607 30064.90361 20797.25454 20106.64763 23075.75919 20365.98566 513001.8396 20638.83746 23649.2834 29344.76887 26826.7331 23241.50115 24558.9413 18890.38544 31039.5341 22522.23797 19136.39006 498748.7566 14472.85897 Asp EIC Results 3662.335785 4347.787233 4583.813687 4203.923077 3334 3182 4256.284942 4528.721112 3918 4911.015217 28168 2718.296788 3421.644581 12155.11512 6705.797197 1454 2236.269006 3674.666291 3960.195702 3085.191925 3243 33897.15629 11989.50018 4013.754717 3994.741025 4002.483707 3013.425367 4072 5499.242766 4173.888452 4778 3045 27344 34 32648 40897.83605 11823.08569 33519.62848 6254.089803 18889.7325 8699.05017 58439.72847 89508.51923 1287.386974 7797.942518 32259.55409 278317.2429 13213.95684 25.93100988 54622.76509 36722.98839 1165 3313.723305 1411 2723 1062 1194 1264 1441.993592 1879 1042.273538 34674.55438 1327 1135 1103 1242 1951 1519 1009 1220 1151 1152 31354.5028 911 1221 1578 1389.318707 1234 1097 951.2824659 1816.250938 1046 1005 29754.16455 65 2HG TIC Results 2379.6125 929937.0141 998440.9202 1069168.264 1766.103125 1957.321316 2171.235413 1099299.67 1892.113359 1145497.368 1622118.682 1975.7625 1905.813369 1984.771547 1546.773382 345366.8361 1970.146877 1600.292662 1295079.348 1979.033494 1900.014468 2014774.842 1280467.562 3027.920939 2127.9625 2185.223102 1412.21632 1225.53659 793902.9575 2112.97766 1011826.018 1652.465949 1557615.714 5827.246875 2067240.808 170291.5777 3583177.525 5628.459375 110893.0043 988477.7222 62002.62615 2617796.438 3029049.197 77559.86208 7178.801639 1450304.234 11398.43176 4855.188278 5736.79375 2501630.235 2308320.5 677076.7539 157407.1728 970.9440275 1329.561653 1737.817176 767532.3147 3001.959482 1516.810698 1431.522293 340394.3165 2295711.772 410910.7765 1573.514574 1086.680241 628545.7785 197697.5771 1267.5 352363.9151 1232.087565 1126.125 387689.2244 2118710.26 1059.402263 939.9966076 1131.44375 1327.84375 1295.81875 1672.782695 714603.4415 2195.4 1634.450019 2509.975414 2056238.225 4077.5625 2HG EIC Results 270 138357.9143 149553.5296 167596.3631 229 191.0320803 227 165613.6082 202 174699.255 247344.8571 269 293 153.6153846 106 46323.90963 188 208 193454.2214 224.7783898 217.5384615 313709.2498 193792 309 186.7152671 208 147 114.9890625 113573.8189 99.34615385 149099.4184 185 237473.5094 350 310272 23652.43084 621035.4005 412.2819881 15248.91958 152807.3429 8894.017817 424871.6039 514037.0952 10228.53963 296.2179893 229797.9352 2742.899876 398.9230769 24.56854897 402789.2198 337960.5409 91317.61085 20168.79702 172 97 187 105992.4709 214 100 174.4615385 43709.26145 333160.3944 52373.78773 229.4774348 95 86368 25699.3888 111 44661.99109 140 219 50886.54115 300712.5488 252 202 79 139 101 88 96216.04266 136 39.28245356 416.0769231 286699.1261 277 Glu TIC Results 6571665.097 2105616.63 2428900.625 2636272.734 5319126.759 4913475.068 6259518.76 2824469.46 6210461.425 3010281.962 492659.6078 5720729.838 5446163.308 7399968.004 6710379.362 325767.923 2744293.793 5883039.872 2628371.845 5277603.5 5635202.765 598050.7795 3618888.413 6774789.104 5235173 7130334.299 5388128.67 6177417.333 3911635.827 6304200.431 2773219.482 5752449.506 494130.8833 2445.575749 608088.0796 5657123.897 5421979.081 6700558.637 2476359.879 5688185.037 2886950.29 899682.6277 7693632.984 1022163.621 2983100.095 7513739.861 15857911.82 3486475.573 3341.736286 861844.5374 782679.0936 402768.302 917124.0403 916524.7667 896398.7647 820508.4048 371541.9572 627541.9594 1423014.712 667199.7068 229728.4523 716686.8442 545835.1978 684639.2191 480477.1016 448996.381 335738.4797 1373593.676 442350.3798 479408.5638 749449.6118 276934.2842 639601.1459 912840.1011 811977.5594 1048159.737 801784.0505 523768.2465 906725.4161 296481.0148 629705.2325 1078039.936 981691.4646 611840.7043 2820.99936 Glu EIC Results 990626.18 275121.6334 327978.707 351267.1984 775707.6208 718882.7063 948702.1825 375655.602 958285.8009 411560.9846 56331.95826 820832.6422 779101.1062 1121221.964 1010130.576 36423.1178 354539.6253 863701.3527 344749.4315 786260.5234 827220.0986 69132.46603 509222.163 1025686.712 767966.1044 1096147.9 796064.9075 934035.5617 553716.6715 967320.9254 379383.6661 856029.8588 57421.82176 173.1177599 68298.64274 837782.48 830819.2785 1052077.06 338847.0227 879267.292 410022.5336 114711.7821 1228190.706 131308.0108 423002.6766 1211303.961 2735000.716 493847.4982 52.31457294 107096.0611 85587.29888 43606.31775 104180.3391 103251.6172 98985.38524 93037.50765 38941.82004 68263.01578 161903.8392 72772.85632 23784.77659 79924.08804 59903.28215 74874.48183 52460.39831 47686.8242 35286.04402 159346.2915 47240.15665 49911.82271 81988.5512 28334.90626 68413.25009 102408 88845.668 120836.1069 89995.74745 56981.70133 102053.4034 30213.95252 68351.40361 122740.3042 110601.1028 66061.18033 129 Ratio of 2HG/Glu TIC 0.000362102 0.44164593 0.411067011 0.405560567 0.000332029 0.000398358 0.000346869 0.38920572 0.000304666 0.380528264 3.292574947 0.000345369 0.000349937 0.000268214 0.000230505 1.060162194 0.000717907 0.000272018 0.492730642 0.000374987 0.000337169 3.368902628 0.353828971 0.00044694 0.000406474 0.000306469 0.000262098 0.00019839 0.202959323 0.00033517 0.364856091 0.000287263 3.152233075 2.38277096 3.399574629 0.030102147 0.660861555 0.000839999 0.04478065 0.17377735 0.02147686 2.909688769 0.393708564 0.075878128 0.00240649 0.193020288 0.000718785 0.001392578 1.716710494 2.902646738 2.949255345 1.681057696 0.17163128 0.001059376 0.001483226 0.002117976 2.065802529 0.004783679 0.001065914 0.002145568 1.481724676 3.203228566 0.752811065 0.002298312 0.002261669 1.399890523 0.588843964 0.000922762 0.796571974 0.002570016 0.001502603 1.39993221 3.312549193 0.001160556 0.001157663 0.001079457 0.001656111 0.002474031 0.001844861 2.410283984 0.003486393 0.001516131 0.002556786 3.360741137 1.445431913 Ratio of 2HG/Glu EIC 0.000272555 0.502897255 0.455985484 0.477119309 0.000295214 0.000265735 0.000239274 0.440865536 0.000210793 0.424479631 4.390844286 0.000327716 0.000376074 0.000137007 0.000104937 1.271827137 0.000530265 0.000240824 0.561144425 0.000285883 0.000262975 4.537799211 0.38056474 0.000301262 0.00024313 0.000189755 0.000184658 0.00012311 0.205111792 0.000102702 0.393004317 0.000216114 4.135596924 2.021745199 4.542872121 0.028232186 0.74749758 0.000391874 0.045002371 0.173789409 0.021691534 3.703818354 0.418531986 0.077897301 0.000700275 0.189711206 0.001002888 0.000807786 0.469631072 3.761008721 3.948723062 2.09413717 0.193595041 0.001665833 0.000979943 0.002009942 2.721816052 0.003134933 0.000617651 0.002397344 1.837699054 4.16846038 0.874305812 0.003064828 0.00181089 1.811150175 0.728315954 0.000696596 0.94542428 0.002804947 0.002671105 1.795895871 4.395530814 0.002460745 0.002273606 0.000653778 0.001544517 0.001772499 0.000862294 3.184490431 0.001989718 0.000320045 0.00376196 4.339903172 2.147286822 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Asp TIC Results Asp EIC Results 2HG TIC Results 2HG EIC Results Glu TIC Results Glu EIC Results Ratio of 2HG/Glu TIC Ratio of 2HG/Glu EIC METABOLITES_END #END