#METABOLOMICS WORKBENCH DAScott_20171122_145837 DATATRACK_ID:1269 STUDY_ID:ST000904 ANALYSIS_ID:AN001471 PROJECT_ID:PR000628 VERSION 1 CREATED_ON November 29, 2017, 3:23 pm #PROJECT PR:PROJECT_TITLE Measurement of 2-hydroxyglutarate for reproducibility project 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 "IDH mutation impairs histone demethylation and results in a block to cell PR:PROJECT_SUMMARY differentiation" by Lu and colleagues, published in Nature in 2012 (Lu et al., PR:PROJECT_SUMMARY 2012). The experiments that will be replicated are those reported in Figures 1B, PR:PROJECT_SUMMARY 2A, 2B, 2D and 4D. Lu and colleagues demonstrated that expression of mutant PR:PROJECT_SUMMARY forms of IDH1 or IDH2 caused global increases in histone methylation and PR:PROJECT_SUMMARY increased levels of 2 hydroxyglutarate (Figure 1B). This was correlated with a PR:PROJECT_SUMMARY block in differentiation (Figures 2A, B and D). This effect appeared to be PR:PROJECT_SUMMARY mediated by the histone demethylase KDM4C (Figure 4D). The Reproducibility PR:PROJECT_SUMMARY Project: Cancer Biology is a collaboration between the Center for Open PR:PROJECT_SUMMARY Scienceand Science Exchange, and the results of the replications will be PR:PROJECT_SUMMARY published by eLife. PR:INSTITUTE Sanford Burnham Prebys Medical Discovery Institute PR:LAST_NAME Scott PR:FIRST_NAME David PR:ADDRESS 10901 N. Torrey Pines Road, La Jolla, California, 92037, USA PR:EMAIL dscott@sbpdiscovery.org PR:PHONE 858-646-3100 #STUDY ST:STUDY_TITLE Measurement of 2-hydroxyglutarate for reproducibility project ST:STUDY_SUMMARY Measurement of D-2-hydroxyglutarate and comparison of peak area with glutamate ST:INSTITUTE Sanford Burnham Prebys Medical Discovery Insititute ST:LAST_NAME David ST:FIRST_NAME Scott ST:ADDRESS 10901 N. Torrey Pines Road ST:EMAIL dscott@sbpdiscovery.org ST:PHONE 858-646-3100 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS 293T cells #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_vec2 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH1wt1 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH1wt2 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH1mut1 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH1mut2 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH2wt2 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run1_IDH2mut2 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH1wt1 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH1wt2 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH1mut1 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH1mut2 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH2wt2 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run2_IDH2mut2 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_vec2 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH1wt1 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH1wt2 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH1mut1 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH1mut2 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH2wt2 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run3_IDH2mut2 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run4_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run4_IDH1wt1 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run4_IDH1mut1 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run4_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run4_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run5_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run5_IDH1wt1 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run5_IDH1mut1 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run5_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run5_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run6_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run6_IDH1wt1 IDH expression:IDH1wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run6_IDH1mut1 IDH expression:IDH1mut SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run6_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 293T Prtcl1_Run6_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run1_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run1_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run1_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run2_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run2_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run2_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run3_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run3_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run3_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run4_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run4_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run4_IDH2mut1 IDH expression:IDH2mut SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run5_vec1 IDH expression:vector SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run5_IDH2wt1 IDH expression:IDH2wt SUBJECT_SAMPLE_FACTORS 3T3_L1 Prtcl2_Run5_IDH2mut1 IDH expression:IDH2mut #COLLECTION CO:COLLECTION_SUMMARY Cells were washed x3 in cold PBS, then scraped into 80% methanol containing 20 CO:COLLECTION_SUMMARY uM L-norvaline CO:COLLECTION_PROTOCOL_FILENAME GC-MS analysis of D-2-hydroxyglutarate CO:SAMPLE_TYPE Adherent cultured mammalian cells #TREATMENT TR:TREATMENT_SUMMARY Cells transfected with empty vector or vector expressing wild-type or mutant TR:TREATMENT_SUMMARY isocitrate dehydrogenase (IDH1 or IDH2; mutations respectively R132H or R172K) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Methanol/water extracts were counter-extracted with chloroform to remove SP:SAMPLEPREP_SUMMARY non-polar metabolites, dried in a centrifugal evaporator, and derivatized in 60 SP:SAMPLEPREP_SUMMARY ul 1:1 mix acetonitrile: MTBSTFA, with heating for 75 min at 75C #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Shimadzu CH:COLUMN_NAME Rxi-5ms #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Shimadzu QP2010 Plus MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples Prtcl1_Run1_vec1 Prtcl1_Run1_vec2 Prtcl1_Run1_IDH1wt1 Prtcl1_Run1_IDH1wt2 Prtcl1_Run1_IDH1mut1 Prtcl1_Run1_IDH1mut2 Prtcl1_Run1_IDH2wt1 Prtcl1_Run1_IDH2wt2 Prtcl1_Run1_IDH2mut1 Prtcl1_Run1_IDH2mut2 Prtcl1_Run2_vec1 Prtcl1_Run2_IDH1wt1 Prtcl1_Run2_IDH1wt2 Prtcl1_Run2_IDH1mut1 Prtcl1_Run2_IDH1mut2 Prtcl1_Run2_IDH2wt1 Prtcl1_Run2_IDH2wt2 Prtcl1_Run2_IDH2mut1 Prtcl1_Run2_IDH2mut2 Prtcl1_Run3_vec1 Prtcl1_Run3_vec2 Prtcl1_Run3_IDH1wt1 Prtcl1_Run3_IDH1wt2 Prtcl1_Run3_IDH1mut1 Prtcl1_Run3_IDH1mut2 Prtcl1_Run3_IDH2wt1 Prtcl1_Run3_IDH2wt2 Prtcl1_Run3_IDH2mut1 Prtcl1_Run3_IDH2mut2 Prtcl1_Run4_vec1 Prtcl1_Run4_IDH1wt1 Prtcl1_Run4_IDH1mut1 Prtcl1_Run4_IDH2wt1 Prtcl1_Run4_IDH2mut1 Prtcl1_Run5_vec1 Prtcl1_Run5_IDH1wt1 Prtcl1_Run5_IDH1mut1 Prtcl1_Run5_IDH2wt1 Prtcl1_Run5_IDH2mut1 Prtcl1_Run6_vec1 Prtcl1_Run6_IDH1wt1 Prtcl1_Run6_IDH1mut1 Prtcl1_Run6_IDH2wt1 Prtcl1_Run6_IDH2mut1 Prtcl2_Run1_vec1 Prtcl2_Run1_IDH2wt1 Prtcl2_Run1_IDH2mut1 Prtcl2_Run2_vec1 Prtcl2_Run2_IDH2wt1 Prtcl2_Run2_IDH2mut1 Prtcl2_Run3_vec1 Prtcl2_Run3_IDH2wt1 Prtcl2_Run3_IDH2mut1 Prtcl2_Run4_vec1 Prtcl2_Run4_IDH2wt1 Prtcl2_Run4_IDH2mut1 Prtcl2_Run5_vec1 Prtcl2_Run5_IDH2wt1 Prtcl2_Run5_IDH2mut1 Factors IDH expression:vector IDH expression:vector IDH expression:IDH1wt IDH expression:IDH1wt IDH expression:IDH1mut IDH expression:IDH1mut IDH expression:IDH2wt IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:IDH2mut IDH expression:vector IDH expression:IDH1wt IDH expression:IDH1wt IDH expression:IDH1mut IDH expression:IDH1mut IDH expression:IDH2wt IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:IDH2mut IDH expression:vector IDH expression:vector IDH expression:IDH1wt IDH expression:IDH1wt IDH expression:IDH1mut IDH expression:IDH1mut IDH expression:IDH2wt IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:IDH2mut IDH expression:vector IDH expression:IDH1wt IDH expression:IDH1mut IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:vector IDH expression:IDH1wt IDH expression:IDH1mut IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:vector IDH expression:IDH1wt IDH expression:IDH1mut IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:vector IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:vector IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:vector IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:vector IDH expression:IDH2wt IDH expression:IDH2mut IDH expression:vector IDH expression:IDH2wt IDH expression:IDH2mut L-glutamic acid 1553494.595 1562635.135 2715159.459 2705972.973 3315413.514 3225586.486 4069710.811 3981932.432 3541029.73 3521472.973 32174445.95 33173521.62 37387854.05 669524.3243 4018556.757 33173521.62 34097886.49 415778.3784 1312451.351 4363089.189 4906618.919 12555597.3 12425705.41 3155270.27 3168751.351 18652443.24 18704972.97 1239713.514 1234521.622 63799932.43 51048481.08 35002643.24 81985983.78 7141408.108 181746421.6 120431208.1 22763681.08 141441454.1 14987764.86 150617324.3 115213051.4 35929856.76 136939213.5 15112213.51 189150462.2 80601170.27 2586600 123825802.7 88606029.73 1798159.459 182459118.9 163871129.7 7186083.784 82287772.97 51463489.19 14419786.49 191570345.9 82942559.46 13915467.57 D-2-hydroxyglutaric acid 4891 4030 31179 10930 116533 124929 49109 48235 4746244 4778555 53258 421863 160253 1076735 6474930 421863 458896 3988146 11779928 61647 32675 65606 85399 2143958 2103946 197851 192021 11387021 12590248 738822 193012 62542544 3574201 40399324 324505 674575 68545648 12152144 74409681 227722 1547759 56088853 3628324 72416259 1093230 469442 39431309 399407 569772 48555618 850754 893896 53355170 1228940 1115177 61027870 3380307 2380821 111581340 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID L-glutamic acid 432 33032 D-2-hydroxyglutaric acid 433 439391 METABOLITES_END #END