Summary of Study ST003319
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 PR002065. The data can be accessed directly via it's Project DOI: 10.21228/M8PR7B 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.
| Study ID | ST003319 |
| Study Title | Changes in the phosphatidylcholine fatty acid composition in subcellular fractions of fibroblasts induced by valinomycin |
| Study Summary | Determination of the impact of valinomycin on the subcellular phosphatidylcholine fatty acid composition of NIH-3T3 fibroblasts challenged with valinomycin for 48 h and disrupted by repeated passage through a syringe. Nuclear, mitochondrial, membrane, and cytosolic fractions were obtained by differential centrifugation. |
| Institute | University of Innsbruck |
| Department | Michael Popp Institute |
| Last Name | Koeberle |
| First Name | Andreas |
| Address | Mitterweg 24, Innsbruck, Tyrol, 6020, Austria |
| andreas.koeberle@uibk.ac.at | |
| Phone | +43 512 507 57903 |
| Submit Date | 2024-07-05 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | wiff |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-01-06 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002065 |
| Project DOI: | doi: 10.21228/M8PR7B |
| Project Title: | Changes in phospholipid fatty acid composition under cytotoxic stress facilitate peroxidation |
| Project Summary: | Programs leading to cell death, such as apoptosis, necroptosis, and ferroptosis, involve an oxidative component linked to lipid metabolism that influences membrane homeostasis. Emerging evidence suggests inter-program cross-talk, emphasizing the need for overarching regulatory mechanisms. We show that under specific cytotoxic stress conditions, exogenous or released polyunsaturated fatty acids (PUFAs) are channeled into overall depleting phospholipids that become vulnerable to peroxidation in the presence of associated redox stress. In fibroblasts, this reprogramming results from reduced growth factor receptor tyrosine kinase (RTK) and phosphatidylinositol-3-kinase (PI3K)/Akt signaling, which reduces de novo fatty acid biosynthesis by mechanisms that differ depending on the specific cytotoxic stressors. We conclude that alterations in PUFA metabolism during cytotoxic stress render cells prone to oxidative modifications in phospholipids. |
| Institute: | University of Innsbruck |
| Department: | Michael Popp Institute |
| Last Name: | Koeberle |
| First Name: | Andreas |
| Address: | Mitterweg 24, Innsbruck, Tyrol, 6020, Austria |
| Email: | andreas.koeberle@uibk.ac.at |
| Phone: | +43 512 507 57903 |
| Funding Source: | Austrian Science Fund (FWF) (P 36299). German Research Council (GRK 1715 and KO 4589/4-1), the Phospholipid Research Center Heidelberg (AKO-2015-037/1-1, AKO-2019-070/2-1, AKO-2O22-100/2-2), the University of Jena (DRM/2013-05 and 2.7-05). |
| Contributors: | André Gollowitzer, Helmut Pein, Konstantin Loeser, Maria Thuermer, and Andreas Koeberle |
Subject:
| Subject ID: | SU003440 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | treatment |
|---|---|---|---|
| SA360234 | 180208_nucleus_Valinomycin | cultured cells | VAL |
| SA360235 | 180222_nucleus_Valinomycin | cultured cells | VAL |
| SA360236 | 180205_nucleus_Valinomycin | cultured cells | VAL |
| SA360237 | 180208_membrane_Valinomycin | cultured cells | VAL |
| SA360238 | 180222_membrane_Valinomycin | cultured cells | VAL |
| SA360239 | 180205_membrane_Valinomycin | cultured cells | VAL |
| SA360240 | 180208_nucleus_DMSO | cultured cells | vehicle |
| SA360241 | 180222_nucleus_DMSO | cultured cells | vehicle |
| SA360242 | 180405_nucleus_DMSO | cultured cells | vehicle |
| SA360243 | 180208_membrane_DMSO | cultured cells | vehicle |
| SA360244 | 180222_membrane_DMSO | cultured cells | vehicle |
| SA360245 | 180405_membrane_DMSO | cultured cells | vehicle |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO003433 |
| Collection Summary: | To separate (peri)nuclear from non-nuclear membranes, NIH-3T3 fibroblasts were suspended in 500 µl ice-cold hypotonic fractionation buffer (10 mM HEPES pH 7.4, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM KCl, 1 mM dithiothreitol) and passed through a 25-gauge needle ten times. After 45 min on ice, the (peri)nuclear (pellet) and non-nuclear fractions (supernatant) were separated by centrifugation (600×g, 10 min, 4°C). Non-nuclear membranes were obtained from the supernatant by centrifugation (100.000×g, 1 h, 4°C). The remaining intact cells in the (peri)nuclear fraction were disrupted by repeated homogenization in fractionation buffer (500 µl). Pellets were washed with PBS pH 7.4 prior to analysis by UPLC-MS/MS and Western Blot. |
| Sample Type: | Fibroblasts |
| Collection Method: | Trypsinization of cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003449 |
| Treatment Summary: | NIH-3T3 fibroblasts were cultivated in DMEM high glucose medium containing heat-inactivated fetal calf serum (FCS, 10%). After cultivation for 24 h, cells were treated with vehicle or VAL (10 µM). |
| Treatment Vehicle: | DMSO |
| Cell Media: | DMEM + 10% FCS |
| Cell Envir Cond: | 37°C, 5% CO2 |
Sample Preparation:
| Sampleprep ID: | SP003447 |
| Sampleprep Summary: | Phospholipids were extracted from cell pellets after subcellular fractionation by successive addition of PBS pH 7.4, methanol, chloroform, and saline to a final ratio of 14:34:35:17. Evaporation of the organic layer yielded a lipid film that was dissolved in methanol, diluted, and subjected to UPLC-MS/MS. |
| Extract Storage: | -20℃ |
Combined analysis:
| Analysis ID | AN005433 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity H-Class |
| Column | Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 5500 QTrap |
| Ion Mode | NEGATIVE |
| Units | relative units |
Chromatography:
| Chromatography ID: | CH004122 |
| Chromatography Summary: | Chromatographic separation of phospholipids was carried out on an Acquity BEH C8 column (1.7 μm, 2.1×100 mm, Waters, Milford, MA) using an Acquity Ultraperformance LC system (Waters). |
| Instrument Name: | Waters Acquity H-Class |
| Column Name: | Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | The gradient was ramped from 70 to 80% B over 5 min and further increased to 100% B within 2 min, followed by isocratic elution for another 2 min. |
| Flow Rate: | 0.75 mL/min |
| Solvent A: | 90% Water/10% Acetonitrile; 10 mM ammonium acetate |
| Solvent B: | 5% Water/95% Acetonitrile; 10 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005159 |
| Analysis ID: | AN005433 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Targeted MRM with pre-optimized settings and subsequent automated integration of selected signals using Analyst 1.6 (Sciex). |
| Ion Mode: | NEGATIVE |
