Summary of Study ST003808
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 PR002382. The data can be accessed directly via it's Project DOI: 10.21228/M8QZ7Q 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 | ST003808 |
| Study Title | Glucose-6-phosphate-dehydrogenase on old peroxisomes maintains self-renewal of epithelial stem cells after asymmetric cell division |
| Study Summary | Peroxisomes play a crucial role in cellular metabolism. Glucose-6-phosphate dehydrogenase (G6PD), the gatekeeper enzyme of the pentose phosphate pathway, is primarily localized in the cytosol. However, studies have reported its presence in peroxisomes as well. This project aims to determine the function of G6PD on the peroxisomal membrane. In this study, we overexpressed G6PD in either the cytosol or on the peroxisomal membrane of mammary epithelial stem-like cells (hMECs). By comparing their lipidomic profiles, we found that peroxisomal membrane-associated G6PD provides NADPH, which feeds into peroxisomal ether lipid synthesis. |
| Institute | University of Helsinki |
| Department | Faculty of Biological and Environmental Sciences |
| Laboratory | Katajisto Laboratory |
| Last Name | Hien |
| First Name | Bui |
| Address | Biocenter 1, Viikinkaari 9 |
| hien.bui@helsinki.fi | |
| Phone | +358294159407 |
| Submit Date | 2025-03-06 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | cdf, mzdata.xml |
| Analysis Type Detail | GC-MS/LC-MS |
| Release Date | 2025-03-24 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002382 |
| Project DOI: | doi: 10.21228/M8QZ7Q |
| Project Title: | Lipidomics of human mammary epithelial stem-like cells (hMECs) overexpressing Glucose-6-phosphate dehydrogenase (G6PD) |
| Project Summary: | Peroxisomes play a crucial role in cellular metabolism. Glucose-6-phosphate dehydrogenase (G6PD), the gatekeeper enzyme of the pentose phosphate pathway, is primarily localized in the cytosol. However, studies have reported its presence in peroxisomes as well. This project aims to determine the function of G6PD on the peroxisomal membrane. To investigate this, we overexpressed G6PD either in the cytosol or on the peroxisomal membrane of human mammary epithelial stem-like cells (hMECs), a cell line that retains characteristics of primary mammary epithelial stem cells, and analyzed their lipidomic profiles for comparison. |
| Institute: | University of Helsinki |
| Department: | Faculty of Biological and Environmental Sciences |
| Laboratory: | Katajisto Laboratory |
| Last Name: | Bui |
| First Name: | Hien |
| Address: | Biocenter 1, Viikinkaari 9, Helsinki, Uusimaa, 00790, Finland |
| Email: | hien.bui@helsinki.fi |
| Phone: | +358294159407 |
Subject:
| Subject ID: | SU003942 |
| 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 | Sample type | G6PD expression |
|---|---|---|---|
| SA417318 | c-neg-2 | cytosolic G6PD | Non-overexpressed |
| SA417319 | c-neg-14 | cytosolic G6PD | Non-overexpressed |
| SA417320 | c-neg-10 | cytosolic G6PD | Non-overexpressed |
| SA417321 | c-neg-6 | cytosolic G6PD | Non-overexpressed |
| SA417322 | c-pos-13 | cytosolic G6PD | Overexpressed |
| SA417323 | c-pos-1 | cytosolic G6PD | Overexpressed |
| SA417324 | c-pos-5 | cytosolic G6PD | Overexpressed |
| SA417325 | c-pos-9 | cytosolic G6PD | Overexpressed |
| SA417326 | p-neg-8 | peroxisomal G6PD | Non-overexpressed |
| SA417327 | p-neg-12 | peroxisomal G6PD | Non-overexpressed |
| SA417328 | p-neg-4 | peroxisomal G6PD | Non-overexpressed |
| SA417329 | p-neg-16 | peroxisomal G6PD | Non-overexpressed |
| SA417330 | p-pos-7 | peroxisomal G6PD | Overexpressed |
| SA417331 | p-pos-11 | peroxisomal G6PD | Overexpressed |
| SA417332 | p-pos-3 | peroxisomal G6PD | Overexpressed |
| SA417333 | p-pos-15 | peroxisomal G6PD | Overexpressed |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO003935 |
| Collection Summary: | Human mammary epithelial cells (hMECs) line FL2 (PMID: 21498687) was used for this study. hMECs were maintained in MEGM mammary epithelial medium (Lonza, CC-3153). G6PD overexpression plasmids were generated in a pRP backbone by inserting the G6PD sequence with or without peroxisomal targeting signal downstream of the hPGK promoter. In addition, the plasmids contain an mCherry sequence downstream of an IRES sequence. mCherry was used as the selection marker for transfected cells in FACS sorting. To over-express G6PD in hMECs, endotoxin-free plasmids were transfected to the cells using jetPRIME (Polyplus, 101000015). 24hours post-transfection, cells were trypsinized, sorted into culture media by Fluorescence-Activated Cell Sorting (FACS), centrifuged at 500g for 5min, washed 1 with cold PBS following by washed 1 with sucrose 0.25M. Pellets were snap freeze with liquid N2 and store at -70. |
| Sample Type: | Epithelial cells |
Treatment:
| Treatment ID: | TR003951 |
| Treatment Summary: | Human mammary epithelial stem like cells (hMECs) were transfected with endotoxin-free G6PD overexpression plasmids using jetPRIME (Polyplus, 101000015). Cells were analyzed 24h post-transfection. |
Sample Preparation:
| Sampleprep ID: | SP003948 |
| Sampleprep Summary: | GC-MS: Fatty acyl (FA) and alkenyl chains, and fatty alcohols (FOHs) were analysed by gas chromatography (GC). Aliquots of cell pellet Folch-extracts were evaporated near to dryness under nitrogen flow and the extracted lipids were transmethylated by heating with 1% H2SO4 in methanol under nitrogen atmosphere. This converts FAs to their methyl esters (FAMEs), phospholipid-derived alkenyl chains to dimethyl acetals (DMAs) and leaves FOHs (1-ol) intact. After adding water, the FAMEs, DMAs and FOHs were extracted with hexane, and the sample solution was dried, and concentrated. LC-MS: Lipids were extracted from cell pellets (at least from one million cells) according to Folch et al (PMID: 13428781), and dissolved in chloroform/methanol 1:2 (v/v). Internal standards [EquiSPLASH® internal standard mixture and Ceramide (Cer) 18:1;O2/17:0, both from Merck] were added and samples were analyzed with LC-MS/MS. |
Chromatography:
| Chromatography ID: | CH004749 |
| Chromatography Summary: | The samples were injected into a GC-2010 Plus gas chromatograph (Shimadzu Scientific Instruments, Kyoto, Japan) with flame-ionization detector (FID) for quantification of the analytes. Both GC systems were equipped with Zebron ZB-wax capillary columns (30 m, 0.25 mm ID and film thickness 0.25 μm; Phenomenex, Torrence CA, USA). GC-FID chromatographic peak areas (in the TXT files; analyte retention times in Table 1) were converted to mol% using the theoretical correction factors for FID (6), and the FOHs were calculated as µmol/10^6 cells. |
| Instrument Name: | Shimadzu GC-2010 |
| Column Name: | Phenomenex Zebron ZB-wax capillary (30m x 0.25mm, 0.25um) |
| Column Temperature: | 280 °C for FID and 210 °C for MSD. |
| Flow Gradient: | NA |
| Flow Rate: | 1.8 ml min–1 for FID and 1.0 ml min–1 for MSD |
| Solvent A: | NA |
| Solvent B: | NA |
| Chromatography Type: | GC |
| Chromatography ID: | CH004750 |
| Chromatography Summary: | The method used acetonitrile/water/isopropanol-based solvent system, Agilent 1290 Infinity HPLC (Agilent Technologies, Santa Clara, CA) equipped with a Luna Omega C18 100 Å (50 x 2.1 mm, 1.6 μm) column (Phenomenex) and Agilent 6490 Triple Quad LC/MS with iFunnel Technology. The lipids species were identified and quantified using lipid class-specific precursor ion and neutral loss detection modes. PE plasmalogens (PE P) were identified with alkenyl chain (16:0, 18:0, 18:1)-specific scans and quantified from MS- scan. - Due to a contamination during the sample run, some samples were removed from analysis |
| Instrument Name: | Agilent 1290 Infinity |
| Column Name: | Phenomenex Luna Omega C18 (50 x 2.1mm, 1.6um, 100 Å) |
| Column Temperature: | 25 |
| Flow Gradient: | 32% B at 0.0 min, 32% B at 1.5 min, 45% B at 4.0 min, 52% B at 5.0 min, 58% B at 8.0 min, 66% B at 11.0 min, 70% B at 14.0 min, 75% B at 18.0 min, 97% B at 21.0 min, 97% B at 25.0 min, 32% B at 26.0 min, 32% B at 32.0 min. flow gradient field is a linear gradient |
| Flow Rate: | 0.200 mL/min |
| Solvent A: | 40% water/60% acetonitrile; 0.1% formic acid; 10mM ammonium formate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 0.1% formic acid; 10mM ammonium formate |
| Chromatography Type: | Reversed phase |
Analysis:
| Analysis ID: | AN006261 |
| Laboratory Name: | Helsinki University Lipidomics Unit (HiLIPID) |
| Analysis Type: | MS |
| Chromatography ID: | CH004749 |
| Num Factors: | 4 |
| Num Metabolites: | 55 |
| Units: | pmol/sample |
| Analysis ID: | AN006262 |
| Analysis Type: | MS |
| Chromatography ID: | CH004750 |
| Num Factors: | 4 |
| Num Metabolites: | 7 |
| Units: | peak area |
