Summary of Study ST002572
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 PR001659. The data can be accessed directly via it's Project DOI: 10.21228/M8872J This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
Study ID | ST002572 |
Study Title | Steady State and Cysteine Flux Metabolomics Study in PTEN WT and PTEN KO MEFs |
Study Type | Quantitative Targeted Mass Spec |
Study Summary | In earlier studies we had indication that the tumor suppressor PTEN was downregulating the cystine glutamate antiporter, xCT; therefore to probe whether this effect on xCT was altering cystine uptake and downstream cystine metabolism, we performed steady state metabolomics via targeted LC-MS/MS on PTEN WT and PTEN KO MEFs. Steady state metabolomics revealed that Pten KO MEFs have a sixfold and fourfold increase in intracellular cystine and cysteine abundance, respectively, as well as a higher abundance of glutathione and the glutathione synthesis intermediate gamma-glutamylcysteine, compared to Pten WT MEFs. In addition to cystine import by xCT as a source of cysteine, cysteine can also be funneled into or recycled from the transsulfuration and choline oxidation pathways. Pten KO MEFs were also found to have increased abundance of transsulfuration pathway metabolites, as well as choline oxidation pathway metabolites. Collectively, this suggests that PTEN regulates cysteine and glutathione metabolism and that PTEN KO cells have more glutathione compared to PTEN WT cells. Next to determine if the increased glutathione in the Pten KO MEFs was being synthesized from increased cystine being brought into the cell by xCT, we performed cystine flux metabolomics via targeted LC-MS/MS on PTEN WT and PTEN KO MEFs and using the heavy isotope 13C2-cystine. Cystine Flux metabolomics revealed Pten KO MEFs were found to have a fourfold and threefold higher accumulation of 13C into intracellular cystine and cysteine, respectively, than Pten WT MEFs, indicating that more extracellular cystine is being brought into the cell by xCT. This result seems plausible given these cells were observed to have higher levels of xCT transporters compared to Pten WT MEFs. Furthermore, there was more cystine flux into glutathione synthesis in Pten KO MEFs, indicated by the sevenfold higher accumulation of heavy isotope labeled glutathione and higher accumulation of its preceding intermediate -glutamylcysteine. Together these findings suggest that PTEN loss heightened the cell’s ability to import cystine via xCT and as a result increased glutathione pools. |
Institute | Mount Sinai |
Department | Oncological Sciences |
Laboratory | Ramon Parsons Laboratory |
Last Name | Cahuzac |
First Name | Kaitlyn |
Address | 6358 Lucent Lane Sandy Springs GA 30328 |
kaitlyncahuzac@gmail.com | |
Phone | 6784537911 |
Submit Date | 2023-04-20 |
Analysis Type Detail | APCI-MS |
Release Date | 2023-04-23 |
Release Version | 1 |
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Combined analysis:
Analysis ID | AN004237 |
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Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Waters Acquity |
Column | Waters XBridge Amide (100 x 4.6mm,3.5um) |
MS Type | APCI |
MS instrument type | QTRAP |
MS instrument name | ABI Sciex 5500 QTrap |
Ion Mode | POSITIVE |
Units | peak area intensity |
Chromatography:
Chromatography ID: | CH003144 |
Instrument Name: | Waters Acquity |
Column Name: | Waters XBridge Amide (100 x 4.6mm,3.5um) |
Column Temperature: | 45 |
Flow Gradient: | 85% buffer B to 30% buffer B from 0 - 3 minutes, then 30% buffer B to 3% buffer B from minute 3 to 12, then 2% buffer B held from minute 12 to 15, followed by 2% buffer B to 85% buffer B from minute 15 to 16, then 85% buffer B held from minute 16-23 in order to re-equilibrate the column. |
Flow Rate: | 400uL/minute |
Solvent A: | 95% water/5% acetonitrile; 20 mM ammonium hydroxide; 20 mM ammonium acetate |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |