Summary of Study ST003407
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 PR002109. The data can be accessed directly via it's Project DOI: 10.21228/M80V5D 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 | ST003407 |
| Study Title | Deficiency in glutathione peroxidase 4 (GPX4) results in abnormal lens development and newborn cataract |
| Study Type | lipidomics |
| Study Summary | This study aims to elucidate the role of GPX4 in lens plasma membrane stability during lens development using in vitro, ex vivo, and in vivo systems. We use lipidomics to analyze the profile of phospholipids and oxidized phospholipids in wild-type and GPX4 KO mice lenses at E18.5. E18.5 lenses were randomly pooled into 4 groups (10mg weight wet/sample) and were subjected to lipid extraction and mass spectrometry analysis. Following data collection, the analysis was conducted using LipidView and the LIPID MAPS® Structure Database (LMSD) software. A notable decrease in the levels of numerous phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acids (PA), and phosphatidylglycerol (PG) species was observed in Gpx4 KO lenses compared to WT. For example, PE 34:1 exhibited a significant decrease (p=0.0479) in Gpx4 KO lenses compared to WT, while PE 36:1 and PE 33:2 displayed a trend of lower levels in Gpx4 KO lenses relative to WT, albeit not statistically significant. Conversely, elevated levels of oxidized phospholipids, particularly oxidized PE, were detected in Gpx4 KO lenses compared to WT. Notably, significant increases were observed in phospholipid oxidation products, including PE(16:0/18:2(O))Na (p=0.0259) and PE(16:0/18:2(OH)(OOH)) (p=0.0401), with a trending increase in PE(16:0/12:1(COOH)(9OH)Na (p=0.0635) in Gpx4 KO lenses relative to WT. These findings strongly indicate that GPX4 deficiency induces lipid peroxidation, consequently altering the composition of plasma membrane lipids. |
| Institute | Augusta University |
| Last Name | Fan |
| First Name | Xingjun |
| Address | 1460 Laney Walker Blvd, Augusta, GA 30912 |
| xfan@augusta.edu | |
| Phone | 7067212019 |
| Submit Date | 2024-08-01 |
| Num Groups | 2 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-11-01 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002109 |
| Project DOI: | doi: 10.21228/M80V5D |
| Project Title: | Deficiency in glutathione peroxidase 4 (GPX4) results in abnormal lens development and newborn cataract |
| Project Type: | Lipidocmics |
| Project Summary: | The human lens consists of a monolayer of lens epithelial cells and extensively elongated fibers that are tightly aligned but separated by the plasma membrane. The integrity of the lens plasma membrane is essential for maintaining lens cellular structure, homeostasis, and transparency. Glutathione peroxidase 4 (GPX4), a selenoenzyme, plays a critical role in protecting against lipid peroxidation. This study aims to elucidate the role of GPX4 in maintaining lens plasma membrane stability during lens development, utilizing in vitro, ex vivo, and in vivo systems. By employing a lipidomics approach, we aim to understand the phospholipid profile of the lens plasma membrane and its alterations following the deletion of GPX4, a key lipid peroxidation detoxification enzyme. Our findings reveal that the deletion of lens-specific GPX4 results in a significant loss of unsaturated phospholipids and an increase in oxidized phospholipids. Consequently, lenses deficient in GPX4 exhibit massive disruption of lens fiber cell structure, significant loss of lens epithelial cells via ferroptosis, and the formation of congenital cataracts. Our study underscores the crucial role of GPX4 in lens development and transparency and offers a potential intervention strategy to prevent lens developmental defects by inhibiting lipid peroxidation. |
| Institute: | Augusta University |
| Last Name: | Fan |
| First Name: | Xingjun |
| Address: | 1460 Laney Walker Blvd, Augusta, GA 30912 |
| Email: | xfan@augusta.edu |
| Phone: | 7067212019 |
| Funding Source: | NEI |
Subject:
| Subject ID: | SU003532 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Sample source |
|---|---|---|---|
| SA372069 | KO-3_ida | Gpx4 Knockout | Eye lens tissue |
| SA372070 | KO-1_283 | Gpx4 Knockout | Eye lens tissue |
| SA372071 | KO-4_ida | Gpx4 Knockout | Eye lens tissue |
| SA372072 | KO-4_283 | Gpx4 Knockout | Eye lens tissue |
| SA372073 | KO-4_253 | Gpx4 Knockout | Eye lens tissue |
| SA372074 | KO-1_253 | Gpx4 Knockout | Eye lens tissue |
| SA372075 | KO-3_283 | Gpx4 Knockout | Eye lens tissue |
| SA372076 | KO-2_ida | Gpx4 Knockout | Eye lens tissue |
| SA372077 | KO-2_283 | Gpx4 Knockout | Eye lens tissue |
| SA372078 | KO-2_253 | Gpx4 Knockout | Eye lens tissue |
| SA372079 | KO-1_ida | Gpx4 Knockout | Eye lens tissue |
| SA372080 | KO-3_253 | Gpx4 Knockout | Eye lens tissue |
| SA372081 | WT-3_283 | Wild type | Eye lens tissue |
| SA372082 | WT-4_ida | Wild type | Eye lens tissue |
| SA372083 | WT-4_283 | Wild type | Eye lens tissue |
| SA372084 | WT-4_253 | Wild type | Eye lens tissue |
| SA372085 | WT-3_ida | Wild type | Eye lens tissue |
| SA372086 | WT-2_283 | Wild type | Eye lens tissue |
| SA372087 | WT-3_253 | Wild type | Eye lens tissue |
| SA372088 | WT-2_ida | Wild type | Eye lens tissue |
| SA372089 | WT-2_253 | Wild type | Eye lens tissue |
| SA372090 | WT-1_ida | Wild type | Eye lens tissue |
| SA372091 | WT-1_283 | Wild type | Eye lens tissue |
| SA372092 | WT-1_253 | Wild type | Eye lens tissue |
| Showing results 1 to 24 of 24 |
Collection:
| Collection ID: | CO003525 |
| Collection Summary: | The eye lens tissues were homogenized with 500 μL of PBS in homogenizing tubes to obtain a homogeneous solution, followed by vortexing for 5 minutes at 4°C. The samples were then centrifuged at 13,000 rpm for 5 min at 4°C using an Eppendorf centrifuge 5810 R. All supernatants were transferred to 1.5 mL microcentrifuge tubes. To each tube, 1.2 mL of methanol and 600 μL of chloroform were added, and the tubes were vortexed for 30 minutes at speed 4 using a Fisher Scientific Multi-tube Vortexer. Subsequently, the samples were centrifuged at 4000 rpm for 10 minutes at 4°C, and the resulting supernatants were collected and transferred to clean 4 mL clear vials. The clear vials were weighed prior to the transfer of supernatants. |
| Collection Protocol Filename: | LCMS_Phospholipids_methodology.pdf |
| Sample Type: | Eye tissue |
Treatment:
| Treatment ID: | TR003541 |
| Treatment Summary: | WT and KO mice lenses |
Sample Preparation:
| Sampleprep ID: | SP003539 |
| Sampleprep Summary: | To separate the organic and non-organic phases in the sample, 1 mL of 0.1M NaCl and 1 mL of chloroform were added. The samples were vortexed for 10 minutes at speed 4 and then centrifuged at 4000 rpm for 10 minutes. The samples were separated into a top layer (non-organic phase) which was discarded, and the lower layer (organic phase) was aliquoted separately using a Pasteur pipette and dried under nitrogen. The dried lipid weight was recorded, and the total weight was subtracted from the weight of the empty vial. The samples were stored at -80°C until ready for LC-MS/MS analysis. Prior to LC-MS/MS analysis, samples were reconstituted with 1 mL of 1:1 chloroform/methanol. Subsequently, 100 μL of sample was transferred into HPLC vials and capped with septa and analyzed by LC-MS/MS. |
| Sampleprep Protocol Filename: | LCMS_Phospholipids_methodology.pdf |
Combined analysis:
| Analysis ID | AN005591 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Shimadzu EXION HPLC |
| Column | Thermo Accucore C18 (100 x 4.6mm, 2um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 5500 QTrap |
| Ion Mode | NEGATIVE |
| Units | counts |
Chromatography:
| Chromatography ID: | CH004249 |
| Chromatography Summary: | See protocol file |
| Methods Filename: | LCMS_Phospholipids_methodology.pdf |
| Instrument Name: | Shimadzu EXION HPLC |
| Column Name: | Thermo Accucore C18 (100 x 4.6mm, 2um) |
| Column Temperature: | 50 |
| Flow Gradient: | 0min, 20% B; 2min, 20% B; 2.1min, 40% B; 10min, 70% B; 10.1min, 100% B; 15min, 100% B; 15.1min, 20% B |
| Flow Rate: | 0.5mL/min |
| Solvent A: | 40% Water/60% acetonitrile |
| Solvent B: | 90% isopropanol/10% acetonitrile; 10mM Ammonium Formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005316 |
| Analysis ID: | AN005591 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | An EMS scan with IDA criteria and precursor scan at 255, and 283 were obtained for each sample, including the SRM 1950 (standard) + Splashmix (internal standard). The results were analyzed from these three scans and provide the oxidized PC or PE with palmitate and stearate, and other phospholipids. The scan range of EMS was from 200 Da to 1000 Da, and for the precursor scan, it was from 400 Da to 1000 Da. The mass spectrometer was operated in negative ionization mode. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | LCMS_Phospholipids_methodology.pdf |
