Summary of Study ST002234

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 PR001424. The data can be accessed directly via it's Project DOI: 10.21228/M8NM68 This work is supported by NIH grant, U2C- DK119886.

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Study IDST002234
Study TitleA metabolic map of the DNA damage response identifies PRDX1 in nuclear ROS scavenging and aspartate synthesis
Study TypeTargetted metabolomics in U2OS PRDX1 WT and PRDX1-/-
Study SummaryTargetted metabolomics in U2OS PRDX1 WT and PRDX1-/- While cellular metabolism impacts the DNA damage response, a systematic understanding of the metabolic requirements that are crucial for DNA damage repair has yet to be achieved. Here, we investigate the metabolic enzymes and processes that are essential when cells are exposed to DNA damage. By integrating functional genomics with chromatin proteomics and metabolomics, we provide a detailed description of the interplay between cellular metabolism and the DNA damage response. Subsequent analysis identified Peroxiredoxin 1, PRDX1, as fundamental for DNA damage repair. During the DNA damage response, PRDX1 translocates to the nucleus where it is required to reduce DNA damage-induced nuclear reactive oxygen species levels. Moreover, PRDX1 controls aspartate availability, which is required for the DNA damage repair-induced upregulation of de novo nucleotide synthesis. Loss of PRDX1 leads to an impairment in the clearance of γΗ2ΑΧ nuclear foci, accumulation of replicative stress and cell proliferation defects, thus revealing a crucial role for PRDX1 as a DNA damage surveillance factor.
Institute
CRG
DepartmentGRSC
LaboratorySdelci_lab
Last NameKourtis
First NameSavvas
AddressCarrer del Dr. Aiguader, 88, 08003 Barcelona, Barcelona, barcelona, 08003, Spain
Emailsavvas.kourtis@crg.eu
Phone653549060
Submit Date2022-07-19
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2023-04-03
Release Version1
Savvas Kourtis Savvas Kourtis
https://dx.doi.org/10.21228/M8NM68
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001424
Project DOI:doi: 10.21228/M8NM68
Project Title:U2OS Etoposide-PRDX1 metabolomics
Project Type:MS quantitative analysis
Project Summary:A metabolic map of the DNA damage response identifies PRDX1 in nuclear ROS scavenging and aspartate synthesis. While cellular metabolism impacts the DNA damage response, a systematic understanding of the metabolic requirements that are crucial for DNA damage repair has yet to be achieved. Here, we investigate the metabolic enzymes and processes that are essential when cells are exposed to DNA damage. By integrating functional genomics with chromatin proteomics and metabolomics, we provide a detailed description of the interplay between cellular metabolism and the DNA damage response. Subsequent analysis identified Peroxiredoxin 1, PRDX1, as fundamental for DNA damage repair. During the DNA damage response, PRDX1 translocates to the nucleus where it is required to reduce DNA damage-induced nuclear reactive oxygen species levels. Moreover, PRDX1 controls aspartate availability, which is required for the DNA damage repair-induced upregulation of de novo nucleotide synthesis. Loss of PRDX1 leads to an impairment in the clearance of γΗ2ΑΧ nuclear foci, accumulation of replicative stress and cell proliferation defects, thus revealing a crucial role for PRDX1 as a DNA damage surveillance factor.
Institute:CRG
Department:GRSC
Laboratory:Sdelci lab
Last Name:Kourtis
First Name:Savvas
Address:Carrer del Dr. Aiguader, 88, 08003 Barcelona
Email:savvas.kourtis@crg.eu
Phone:653549060

Subject:

Subject ID:SU002320
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

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

mb_sample_id local_sample_id Genotype Treatment
SA212931109773PRDX1 KO Etop-0h
SA212932109774PRDX1 KO Etop-0h
SA212933109775PRDX1 KO Etop-0h
SA212934109779PRDX1 KO Etop-24h
SA212935109780PRDX1 KO Etop-24h
SA212936109781PRDX1 KO Etop-24h
SA212937109778PRDX1 KO Etop-8h
SA212938109777PRDX1 KO Etop-8h
SA212939109776PRDX1 KO Etop-8h
SA212940109772PRDX1 KO Untreated
SA212941109770PRDX1 KO Untreated
SA212942109771PRDX1 KO Untreated
SA212943109755WT Etop-0h
SA212944109757WT Etop-0h
SA212945109756WT Etop-0h
SA212946109762WT Etop-24h
SA212947109763WT Etop-24h
SA212948109761WT Etop-24h
SA212949109758WT Etop-8h
SA212950109760WT Etop-8h
SA212951109759WT Etop-8h
SA212952109764WT Noco-18h
SA212953109765WT Noco-18h
SA212954109766WT Noco-18h
SA212955109767WT Noco-23h
SA212956109769WT Noco-23h
SA212957109768WT Noco-23h
SA212958109753WT Untreated
SA212959109752WT Untreated
SA212960109754WT Untreated
Showing results 1 to 30 of 30

Collection:

Collection ID:CO002313
Collection Summary:For metabolite collection, plates containing 0.2-0.4 million cells per well were gently washed with 75mM ammonium carbonate buffer pH 7.4 at room temperature, transferred on ice, and metabolites were extracted with 80:20 ice-cold MeOH:H2O solution. Cells were scraped off and samples were collected in tubes, then snap frozen in liquid nitrogen to stop all metabolic reactions.
Sample Type:U2OS cells

Treatment:

Treatment ID:TR002332
Treatment Summary:U2-OS cells were seeded in 6 well plates. Etoposide treatment (1μM for 3 hours) was performed at different times to be able to terminate the experiment and extract the metabolites simultaneously for all samples. At the last timepoint – treatment for the no release samples – the medium was changed in all wells in order to have growth medium of the same composition at the time of metabolite extraction. Each sample was prepared in triplicates.

Sample Preparation:

Sampleprep ID:SP002326
Sampleprep Summary:Once all wells have been collected, samples were thawed and centrifuged in a table-top centrifuge at maximum speed at 4 degrees. Supernatants containing metabolites were transferred into HPLC vial and stored at -80 degrees until processing by the metabolomics facility (Pro-Met, CeMM). Cleared extracts were dried under nitrogen. Samples were taken up in MS-grade water and mixed with the heavy isotope labeled internal standard mix.

Combined analysis:

Analysis ID AN003644
Analysis type MS
Chromatography type Reversed phase
Chromatography system Agilent 1290 Infinity II
Column Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name Agilent 6470 TQ
Ion Mode NEGATIVE
Units au

Chromatography:

Chromatography ID:CH002698
Chromatography Summary:A 1290 Infinity II UHPLC system (Agilent Technologies) coupled with a 6470 triple quadrupole mass spectrometer (Agilent Technologies) was used for the LC-MS/MS analysis. The chromatographic separation for samples was carried out on a ZORBAX RRHD Extend-C18, 2.1 x 150 mm, 1.8 µm analytical column (Agilent Technologies). The column was maintained at a temperature of 40°C and 4 µL of sample was injected per run. The mobile phase A was 3% methanol (v/v), 10 mM tributylamine, 15 mM acetic acid in water and mobile phase B was 10 mM tributylamine, 15 mM acetic acid in methanol. The gradient elution with a flow rate of 0.25 mL/min was performed for a total time of 24 min. Afterwards back-flushing of the column using a 6port/2-position divert valve was carried out for 8 min using acetonitrile, followed by 8 min of column equilibration with 100% mobile phase A. The triple quadrupole mass spectrometer was operated in negative electrospray ionization mode, spray voltage 2 kV, gas temperature 150 °C, gas flow 1.3 L/min, nebulizer 45 psi, sheath gas temperature 325 °C, sheath gas flow 12 L/min. The metabolites of interest were detected using a dynamic MRM mode.
Instrument Name:Agilent 1290 Infinity II
Column Name:Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
Column Temperature:40
Flow Gradient:The gradient elution with a flow rate of 0.25 mL/min was performed for a total time of 24 mi
Flow Rate:0.25ml/min
Solvent A:3% methanol/97% water; 15 mM acetic acid; 10 mM tributylamine
Solvent B:100% methanol; 15 mM acetic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003395
Analysis ID:AN003644
Instrument Name:Agilent 6470 TQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:The chromatographic separation for samples was carried out on a ZORBAX RRHD Extend-C18, 2.1 x 150 mm, 1.8 µm analytical column (Agilent Technologies). The column was maintained at a temperature of 40°C and 4 µL of sample was injected per run. The mobile phase A was 3% methanol (v/v), 10 mM tributylamine, 15 mM acetic acid in water and mobile phase B was 10 mM tributylamine, 15 mM acetic acid in methanol. The gradient elution with a flow rate of 0.25 mL/min was performed for a total time of 24 min. Afterwards back-flushing of the column using a 6port/2-position divert valve was carried out for 8 min using acetonitrile, followed by 8 min of column equilibration with 100% mobile phase A. The triple quadrupole mass spectrometer was operated in negative electrospray ionization mode, spray voltage 2 kV, gas temperature 150 °C, gas flow 1.3 L/min, nebulizer 45 psi, sheath gas temperature 325 °C, sheath gas flow 12 L/min. The metabolites of interest were detected using a dynamic MRM mode.
Ion Mode:NEGATIVE
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