Summary of Study ST002791

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 PR001739. The data can be accessed directly via it's Project DOI: 10.21228/M8XD9T This work is supported by NIH grant, U2C- DK119886.

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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 IDST002791
Study TitleNucleotide metabolism in pancreatic cancer cells
Study SummaryThe Experiment analyzes the cancer cell metabolism in two pancreatic cancer cell lines, (i.e. Panc02 and KPC FC 1245) after the knockdown of the protein cytidine deaminase (CDA). Murine Panc02 and KPC FC1245 pancreatic cancer cell lines were genetically engineered using a doxycycline inducible CRISPR/Cas9 platform with a target specific gRNA for CDA and a control non-targeting NT gRNA. CDA knockdown cells show a significant decrease of intracellular uridine levels and the accumulation of intracellular cytidine. Consistent with the decrease in intracellular uridine, sgCda cells show reduced intracellular levels of UMP, UDP and UTP compared to sgNT cells while we see no change in adenine and cytosine nucleotides (i.e., AMP, ADP, ATP and CMP, CDP, CTP, respectively) or in UDP-hexose.
Institute
VIB-KU Leuven
Last NameMazzone
First NameMassimiliano
AddressCampus Gasthuisberg Herestraat 49, box 912 B-3000 Leuven Belgium
Emailmassimiliano.mazzone@kuleuven.vib.be
Phone+32-16-37.32.13
Submit Date2023-07-16
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2024-01-24
Release Version1
Massimiliano Mazzone Massimiliano Mazzone
https://dx.doi.org/10.21228/M8XD9T
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001739
Project DOI:doi: 10.21228/M8XD9T
Project Title:Nucleotide metabolism in pancreatic cancer cells
Project Summary:The Experiment analyzes the cancer cell metabolism in two pancreatic cancer cell lines, (i.e. Panc02 and KPC FC 1245) after the knockdown of the protein cytidine deaminase (CDA). Murine Panc02 and KPC FC1245 pancreatic cancer cell lines were genetically engineered using a doxycycline inducible CRISPR/Cas9 platform with a target specific gRNA for CDA and a control non-targeting NT gRNA. CDA knockdown cells show a significant decrease of intracellular uridine levels and the accumulation of intracellular cytidine. Consistent with the decrease in intracellular uridine, sgCda cells show reduced intracellular levels of UMP, UDP and UTP compared to sgNT cells while we see no change in adenine and cytosine nucleotides (i.e., AMP, ADP, ATP and CMP, CDP, CTP, respectively) or in UDP-hexose.
Institute:VIB Center for Cancer Biology
Last Name:Mazzone
First Name:Massimiliano
Address:Herestraat 49, box 912, Leuven, Flemish Brabant, 3000, Belgium
Email:massimiliano.mazzone@kuleuven.be
Phone:+32-16-37.32.13

Subject:

Subject ID:SU002898
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Not applicable

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Cell Line
SA299530KPC_CDAKD_1sgCDA KPC_FC1245
SA299531KPC_CDAKD_3sgCDA KPC_FC1245
SA299532KPC_CDAKD_4sgCDA KPC_FC1245
SA299533KPC_CDAKD_2sgCDA KPC_FC1245
SA299534Panc02_CDAKD_3sgCDA Panc02
SA299535Panc02_CDAKD_4sgCDA Panc02
SA299536Panc02_CDAKD_2sgCDA Panc02
SA299537Panc02_CDAKD_1sgCDA Panc02
SA299538KPC_NT_2sgNT KPC_FC1245
SA299539KPC_NT_1sgNT KPC_FC1245
SA299540KPC_NT_3sgNT KPC_FC1245
SA299541KPC_NT_4sgNT KPC_FC1245
SA299542Panc02_NT_4sgNT Panc02
SA299543Panc02_NT_1sgNT Panc02
SA299544Panc02_NT_2sgNT Panc02
SA299545Panc02_NT_3sgNT Panc02
Showing results 1 to 16 of 16

Collection:

Collection ID:CO002891
Collection Summary:On day 2, cells were harvested on ice by washing them once in ice-cold saline solution (9 g/l NaCl). Cells were covered with 250 μl of pre-cooled 80% methanol for 2 minutes scraped, transferred to fresh vials, and precipitated overnight at -80°C. Then they were centrifuged at 20.009 g for 15min, 4 degrees C and supernatant transferred to an M/S vial.
Sample Type:Cultured cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002907
Treatment Summary:Cancer cells were cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep in 6-well plates. On day 1, the cells and empty wells (for background noise control), were washed with PBS, and replenished with DMEM containing 10% FBS, 1% Pen/Strep, 5.5 mM glucose, 2 mM glutamine, 1% Pen/Strep, 0.1 mM cytidine (Sigma-Aldrich), or alternately, 0.1 mM labelled cytidine (13C9, 98% 15N3, 96-98%, Eurisotop) for 48h.

Sample Preparation:

Sampleprep ID:SP002904
Sampleprep Summary:The samples were centrifuged at 20.000 x g for 15 min at 4°C, supernatant was transferred to appropriate M/S vial for analysis. The cell pellet was dissolved in 100 μl of 200 mM NaOH (for 20 minutes at 95°C), and the protein concentration was determined by using bicinchoninic acid (BCA) reagent method.
Processing Storage Conditions:-80℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN004541
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity
Column Waters Acquity UPLC HSS T3 (150 x 2.1mm, 1.8um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE
Units AUC

Chromatography:

Chromatography ID:CH003411
Chromatography Summary:C18 IP REVERSE PHASE
Instrument Name:Waters Acquity
Column Name:Waters Acquity UPLC HSS T3 (150 x 2.1mm, 1.8um)
Column Temperature:40
Flow Gradient:The gradient started with 5% solvent B and 95% solvent A and remained at 5% B until 2min post injection. A linear gradient to 37% B was carried out until 7min and increased to 41% until 14min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min.
Flow Rate:0.25 mL/min
Solvent A:100% water; 10mM tributylamine; 15mM acetic acid
Solvent B:100% methanol
Chromatography Type:Reversed phase

MS:

MS ID:MS004288
Analysis ID:AN004541
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:EI-Maven polly, ThermoFisher Xcalibur, Metabolites were annotated using the inhouse standard metabolite library- elution time and m/z values.
Ion Mode:NEGATIVE
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