Summary of Study ST000548

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000402. The data can be accessed directly via it's Project DOI: 10.21228/M83890 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000548
Study TitleReplication study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate
Study SummaryThe Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate” by Ward and colleagues, published in Cancer Cell in 2010 (Ward et al., 2010). The experiments that will be replicated are those reported in Figures 2, 3 and 5. Ward and colleagues demonstrate the mutations in isocitrate dehydrogenase 2 (IDH2), commonly found in acute myeloid leukemia (AML), abrogate the enzyme’s wild-type activity and confer to the mutant neomorphic activity that produces the oncometabolite 2-hydroxyglutarate (2-HG) (Figures 2 and 3). They then show that elevated levels of 2-HG are correlated with mutations in IDH1 and IDH2in AML patient samples (Figure 5). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published by eLife.
Institute
University of California, Davis
DepartmentGenome and Biomedical Sciences Facility
LaboratoryWCMC Metabolomics Core
Last NameFiehn
First NameOliver
AddressHealth Sciences Drive, Davis, California, 95616, USA
Emailofiehn@ucdavis.edu
Phone(530) 754-8258
Submit Date2017-01-06
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailGC-MS
Release Date2017-07-10
Release Version1
Oliver Fiehn Oliver Fiehn
https://dx.doi.org/10.21228/M83890
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR000402
Project DOI:doi: 10.21228/M83890
Project Title:Reproducibility study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate
Project Summary:The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate” by Ward and colleagues, published in Cancer Cell in 2010 (Ward et al., 2010). The experiments that will be replicated are those reported in Figures 2, 3 and 5. Ward and colleagues demonstrate the mutations in isocitrate dehydrogenase 2 (IDH2), commonly found in acute myeloid leukemia (AML), abrogate the enzyme’s wild-type activity and confer to the mutant neomorphic activity that produces the oncometabolite 2-hydroxyglutarate (2-HG) (Figures 2 and 3). They then show that elevated levels of 2-HG are correlated with mutations in IDH1 and IDH2in AML patient samples (Figure 5). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published by eLife.
Institute:University of California, Davis
Department:Genome and Biomedical Sciences Facility
Laboratory:WCMC Metabolomics Core
Last Name:Fiehn
First Name:Oliver
Address:Health Sciences Drive, Davis, California, 95616, USA
Email:ofiehn@ucdavis.edu
Phone:(530) 754-8258
Funding Source:NIH U24DK097154

Subject:

Subject ID:SU000570
Subject Type:Cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

Factors:

Subject type: Cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Time Point Sample Source
SA028240Blank003Blank -
SA028241Blank001Blank -
SA028242Blank002Blank -
SA028243MUT03CS-24h 293T HEK cell line
SA028244WT08CS-24h 293T HEK cell line
SA028245MUT05CS-24h 293T HEK cell line
SA028246WT09CS-24h 293T HEK cell line
SA028247V05CS-24h 293T HEK cell line
SA028248V06CS-24h 293T HEK cell line
SA028249V03CS-24h 293T HEK cell line
SA028250V08CS-24h 293T HEK cell line
SA028251MUT02CS-24h 293T HEK cell line
SA028252V01CS-24h 293T HEK cell line
SA028253WT01CS-24h 293T HEK cell line
SA028254V04CS-24h 293T HEK cell line
SA028255V10CS-24h 293T HEK cell line
SA028256MUT01CS-24h 293T HEK cell line
SA028257WT02CS-24h 293T HEK cell line
SA028258WT07CS-24h 293T HEK cell line
SA028259WT05CS-24h 293T HEK cell line
SA028260MUT06CS-24h 293T HEK cell line
SA028261V09CS-24h 293T HEK cell line
SA028262WT10CS-24h 293T HEK cell line
SA028263MUT09CS-24h 293T HEK cell line
SA028264MUT04CS-24h 293T HEK cell line
SA028265MUT07CS-24h 293T HEK cell line
SA028266V02CS-24h 293T HEK cell line
SA028267WT03CS-24h 293T HEK cell line
SA028268V07CS-24h 293T HEK cell line
SA028269MUT10CS-24h 293T HEK cell line
SA028270MUT08CS-24h 293T HEK cell line
SA028271WT06CS-24h 293T HEK cell line
SA028272WT04CS-24h 293T HEK cell line
SA028273CS56_V06_48CS-48h 293T HEK cell line
SA028274CS53_V03_48CS-48h 293T HEK cell line
SA028275CS48_MUT08_48CS-48h 293T HEK cell line
SA028276CS42_MUT02_48CS-48h 293T HEK cell line
SA028277CS37_WT07_48CS-48h 293T HEK cell line
SA028278CS49_MUT09_48CS-48h 293T HEK cell line
SA028279CS43_MUT03_48CS-48h 293T HEK cell line
SA028280CS51_V01_48CS-48h 293T HEK cell line
SA028281CS52_V02_48CS-48h 293T HEK cell line
SA028282CS41_MUT01_48CS-48h 293T HEK cell line
SA028283CS44_MUT04_48CS-48h 293T HEK cell line
SA028284CS33_WT03_48CS-48h 293T HEK cell line
SA028285CS58_V08_48CS-48h 293T HEK cell line
SA028286CS47_MUT07_48CS-48h 293T HEK cell line
SA028287CS59_V09_48CS-48h 293T HEK cell line
SA028288CS46_MUT06_48CS-48h 293T HEK cell line
SA028289CS35_WT05_48CS-48h 293T HEK cell line
SA028290CS32_WT02_48CS-48h 293T HEK cell line
SA028291CS60_V10_48CS-48h 293T HEK cell line
SA028292CS39_WT09_48CS-48h 293T HEK cell line
SA028293CS31_WT01_48CS-48h 293T HEK cell line
SA028294CS54_V04_48CS-48h 293T HEK cell line
SA028295CS55_V05_48CS-48h 293T HEK cell line
SA028296CS34_WT04_48CS-48h 293T HEK cell line
SA028297CS45_MUT05_48CS-48h 293T HEK cell line
SA028298CS36_WT06_48CS-48h 293T HEK cell line
SA028299CS50_MUT10_48CS-48h 293T HEK cell line
SA028300CS38_WT08_48CS-48h 293T HEK cell line
SA028301CS40_WT10_48CS-48h 293T HEK cell line
SA028302CS57_V07_48CS-48h 293T HEK cell line
SA028303HR_08_IDH2HR Peripheral blood mono-nuclear cells
SA028304HR_07_IDH2HR Peripheral blood mono-nuclear cells
SA028305HR_10_IDH1HR Peripheral blood mono-nuclear cells
SA028306HR_05_IDH2HR Peripheral blood mono-nuclear cells
SA028307HR_02_WTHR Peripheral blood mono-nuclear cells
SA028308HR_09_IDH1HR Peripheral blood mono-nuclear cells
SA028309HR_11_IDH1HR Peripheral blood mono-nuclear cells
SA028310HR_12_IDH1HR Peripheral blood mono-nuclear cells
SA028311HR_04_WTHR Peripheral blood mono-nuclear cells
SA028312HR_01_WTHR Peripheral blood mono-nuclear cells
SA028313HR_03_WTHR Peripheral blood mono-nuclear cells
SA028314HR_06_IDH2HR Peripheral blood mono-nuclear cells
SA028315QC002QC -
SA028316QC003QC -
SA028317QC011QC -
SA028318QC004QC -
SA028319QC005QC -
SA028320QC06QC -
SA028321QC008QC -
SA028322QC007QC -
SA028323QC010QC -
SA028324QC001QC -
SA028325QC009QC -
Showing results 1 to 86 of 86

Collection:

Collection ID:CO000564
Collection Summary:293T Cells were harvested 24 and 48 hours after transfection by removal of media and rapid quenching with 1.5 mL per 10 cm plate of -80°C methanol. Cells were incubated at -80°C for 15 minutesmin, then scraped off the dish and centrifuged for 5 min at 2,000 x g at 4°C to pellet cellular debris. Pellet was re-extracted by addition of 500 µL of -80°C 80% methanol in water, vortexed, then incubated at 4°C for 15 minutes and centrifuged for 5 minutes at 2,000 x g at 4°C. Peripheral blood mono-nuclear cells were centrifuged at 2,000 x g, freeze media removed and extracted using same procedure as 293T cells.
Collection Protocol Filename:The common feature of IDH1 and IDH2 mutations.pdf
Sample Type:Cells
Collection Location:UC Davis Genome and Biomedical Sciences Facility

Treatment:

Treatment ID:TR000584
Treatment Summary:239T cells (ATCC CRL-3216) were grown in DMEM (Invitrogen) with 10% FBS (Hyclone) at -37°C in 10% CO2 and transfected with pcDNA3.1, pcDNA3.1-IDH2WT, pcDNA3.1-IDH2R172K (Invitrogen and Origene) with Lipofectamine 2000 (Invitrogen) according to manufacturer instructions. 6x10^5 cells were seeded in 6 well plates for protocol 1 and 3.5x10^6 cells were seeded in 10 cm plates for protocol 2. Identity of all vectors was confirmed by sequencing and vector integrity with agarose gel electrophoresis.The common feature of IDH1 and IDH2 mutations.pdf
Treatment Protocol Filename:239T cells (ATCC CRL-3216) were grown in DMEM (Invitrogen) with 10% FBS (Hyclone) at 37°C in 10% CO2 and transfected with pcDNA3.1, pcDNA3.1-IDH2WT, pcDNA3.1-IDH2R172K (Invitrogen and Origene) with Lipofectamine 2000 (Invitrogen) according to manufacturer instructions.
Cell Harvesting:24-48 hours after transfection

Sample Preparation:

Sampleprep ID:SP000577
Sampleprep Summary:Supernatants were combined and evaporated to dryness. Samples were then re-suspended in 200 µL LC-MS-grade water. A 2 mL AG-1 X8 100-200 anion exchange resin (Bio-Rad) was washed with 5 column volumes (10 mL) of 3 N HCl followed by transfer of re-suspended extracts to resin column. Metabolites were eluted using 10 mL 3 N HCl. Samples were evaporated to dryness and re-suspended in 100 µL MTBSTFA/ACN (1:1, v/v) and shaken at 60°C for 1 hour. Derivatized extracts were further diluted (1:4) with a MTBSTFA/ACN (1:1, v/v) mixture and transferred to glass vials with micro-inserts and capped immediately.
Sampleprep Protocol Filename:The common feature of IDH1 and IDH2 mutations.pdf

Combined analysis:

Analysis ID AN000836
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890A
Column Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent
Ion Mode POSITIVE
Units Counts

Chromatography:

Chromatography ID:CH000597
Methods Filename:Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf
Instrument Name:Agilent 7890A
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Column Temperature:250°C
Flow Rate:1mL/min
Sample Injection:0.2µl
Oven Temperature:100°C (3 min), 4°C/min to 230°C (hold 4 min), 30°C/min to 300°C (hold 5 min)
Transferline Temperature:230°C
Randomization Order:Excel generated
Chromatography Type:GC

MS:

MS ID:MS000737
Analysis ID:AN000836
Instrument Name:Agilent
Instrument Type:Single quadrupole
MS Type:EI
Ion Mode:POSITIVE
Ion Source Temperature:230°C
Ionization Energy:70eV
Mass Accuracy:Nominal
Scan Range Moverz:50-600 Da
Scanning Cycle:2.71 scans/sec
Scanning Range:50-600 Da
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