Summary of study ST001658

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 PR001063. The data can be accessed directly via it's Project DOI: 10.21228/M89695 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001658
Study TitleControl of Topoisomerase II Activity and Chemotherapeutic Inhibition by TCA Cycle Metabolites
Study SummaryTopoisomerase II (topo II) is essential for disentangling newly-replicated chromosomes. DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapies to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our works reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with important ramifications for the clinical use of anti-topo II therapies.
Institute
Johns Hopkins University
Last NameMosher
First NameEric
Address725 North Wolfe Street, Biophysics 307
Emailemosher2@jhmi.edu
Phone410-952-9154
Submit Date2021-01-21
Raw Data AvailableYes
Raw Data File Type(s).mzXML
Analysis Type DetailLC-MS
Release Date2021-02-17
Release Version1
Eric Mosher Eric Mosher
https://dx.doi.org/10.21228/M89695
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001063
Project DOI:doi: 10.21228/M89695
Project Title:Control of Topoisomerase II Activity and Chemotherapeutic Inhibition by TCA Cycle Metabolites
Project Summary:Topoisomerase II (topo II) is essential for disentangling newly-replicated chromosomes. DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapies to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our works reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with important ramifications for the clinical use of anti-topo II therapies.
Institute:Johns Hopkins University
Last Name:Mosher
First Name:Eric
Address:725 North Wolfe Street, Biophysics 307
Email:emosher2@jhmi.edu
Phone:410-952-9154

Subject:

Subject ID:SU001735
Subject Type:Yeast
Subject Species:Saccharomyces cerevisiae
Taxonomy ID:4932
Cell Strain Details:BY4741

Factors:

Subject type: Yeast; Subject species: Saccharomyces cerevisiae (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA151945F4HPLC fraction 4
SA151946F5HPLC fraction 5
SA151947F6HPLC fraction 6
Showing results 1 to 3 of 3

Collection:

Collection ID:CO001728
Collection Summary:Yeast cultures were grown in SC-sulfate media and were harvested at OD600 0.4-0.8 by vacuum filtration then washed with 100mM ammonium acetate - TEA (pH 8.0). Metabolites were extracted by the addition of pre-chilled 10mL 90% methanol, 10mM ammonium acetate - TEA (pH 8.0). Cell debris was removed by centrifugation at 4000g for 10-15 minutes. Extracts were subsequently passed through 3 kDa MWCO filters. Filtrate was diluted 2-fold with water and lyophilized. Lyophilized metabolite extracts were resuspended with water and pH was adjusted to 10 with TEA. The extract was then passed through a mixed-mode anion exchange solid phase extraction cartridge and fractionated by reverse-phase HPLC on a C18 column. One column volume of water plus 0.1% formic acid and eight column volumes of methanol were passed through the column. Fractions that eluted during the aqueous phase were then lyophilized.
Sample Type:Yeast cells

Treatment:

Treatment ID:TR001748
Treatment Summary:Active fractions were run on a normal-phase HPLC on an amide column and fractions were collected. Fractions 4, 5, and 6 were subjected to metabolomics analyses.

Sample Preparation:

Sampleprep ID:SP001741
Sampleprep Summary:Fractions of interest were lyophilized then reconstituted in water plus 0.1% formic acid such that samples reached a final concentration of 20 mg/mL.

Combined analysis:

Analysis ID AN002706 AN002707 AN002708 AN002709
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase Normal phase Normal phase
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column Thermo Hypersil Gold aQ (150 x 2.1mm, 1.9um) Thermo Hypersil Gold aQ (150 x 2.1mm, 1.9um) PolyLC Polyhydroxyethyl A (200 x 2.1mm, 5um) PolyLC Polyhydroxyethyl A (200 x 2.1mm, 5um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE
Units Peak Area Peak Area Peak Area Peak Area

Chromatography:

Chromatography ID:CH001997
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:Thermo Hypersil Gold aQ (150 x 2.1mm, 1.9um)
Column Temperature:30
Flow Gradient:0-2min 0% B, 2-13min 0-60% B, 13-15min 60-90% B, 15-16min 90-0% B, 16-20min 0% B
Flow Rate:0.2 mL/min
Solvent A:1mM ammonium acetate + 0.1% formic acid in water
Solvent B:1mM ammonium acetate + 0.1% formic acid in 20/80 water/acetonitrile
Chromatography Type:Reversed phase
  
Chromatography ID:CH001998
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:PolyLC Polyhydroxyethyl A (200 x 2.1mm, 5um)
Column Temperature:30
Flow Gradient:0-2min 80% B, 2-13min 80-40% B, 13-15min 40-10% B, 15-16min 10-80% B, 16-20min 80% B
Flow Rate:0.2 mL/min
Solvent A:1mM ammonium acetate + 0.1% formic acid in water
Solvent B:1mM ammonium acetate + 0.1% formic acid in 20/80 water/acetonitrile
Chromatography Type:Normal phase

MS:

MS ID:MS002503
Analysis ID:AN002706
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software.
Ion Mode:POSITIVE
  
MS ID:MS002504
Analysis ID:AN002707
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software.
Ion Mode:NEGATIVE
  
MS ID:MS002505
Analysis ID:AN002708
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software.
Ion Mode:POSITIVE
  
MS ID:MS002506
Analysis ID:AN002709
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS1 data was acquired at a resolution of 140,000 over a scan range of 65-850 m/z. Data was analyzed with Thermo Compound Discoverer software.
Ion Mode:NEGATIVE
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