Summary of Study ST002422
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 PR001559. The data can be accessed directly via it's Project DOI: 10.21228/M85X3W 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 | ST002422 |
| Study Title | UBXD8 lipidomics from whole cells (Part 2) |
| Study Summary | The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipids found in mitochondria-associated membranes (MAM). LC-MS/MS lipidomics found significant changes in distinct lipid species in the MAM fraction of UBXD8 knockout cells. Our results suggest that lipids in MAM are regulated by UBXD8. |
| Institute | University of Arizona |
| Department | Immunobiology |
| Laboratory | Purdy Lab |
| Last Name | Purdy |
| First Name | John |
| Address | PO Box 245221, Tucson, Arizona, 85724, USA |
| purdylab@gmail.com | |
| Phone | 520-626-4371 |
| Submit Date | 2023-01-01 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-01-16 |
| Release Version | 1 |
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Collection:
| Collection ID: | CO002504 |
| Collection Summary: | Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and streptomycin. Cells were maintained in a humidified, 5 % CO2 atmosphere at 37°C. The CRISPR-Cas9 gene editing system was used to generate UBXD8 knockout cell lines in HEK293T cells. Cells were seeded into four 150 mm TC dishes. Cells were lysed in Homogenization buffer (225 mM mannitol, 75 mM sucrose, and 30 mM Tris-Cl, pH 7.4) using a Dounce homogenizer. The lysate was centrifuged three times at 600xg for 5 minutes to remove unlysed cells and nuclei resulting in post-nuclear supernatants (PNS). The cleared lysate was centrifuged at 7000xg to separate crude mitochondrial pellet and supernatant containing microsomes. The supernatant was cleared by centrifugation at 20,000xg for 30 minutes followed by microsome isolation using high-speed centrifugation at 100,000xg for 1 hour. The crude mitochondrial pellet was washed twice in homogenization buffer containing 0.1 mM EGTA at 7000xg and 10,000xg for 10 minutes. MAMs were isolated from crude mitochondria using 30 % Percoll gradient centrifugation at 95,000xg for 1 hr in a swinging-bucket rotor. The banded MAM fraction was washed once with phosphate-buffered saline (PBS) before lysing in lysis buffer (50 mM Tris-Cl, pH 7.2, 150 mM NaCl). The pure mitochondrial fractions were resuspended and washed in mitochondrial resuspension buffer (250 mM mannitol, 0.5 mM EGTA, 5 mM HEPES pH7.4). Protein concentrations for both soluble and pellet fractions were determined by DC protein assay kit (Biorad). For lipidomics of MAMs, the final banded MAM fraction was washed twice with liquid chromatography-mass spectrometry (LC-MS) grade PBS and the final MAM pellet was resuspended in LC-MS grade PBS. Next, 1mL of cold 50% methanol was added and the MAMS were transferred to glass vials. Chloroform was added (0.5mL) and the mixture was gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration. Extracted lipids were resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. |
| Sample Type: | Epithelial cells |
