Summary of Study ST003424
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 PR002115. The data can be accessed directly via it's Project DOI: 10.21228/M87C0V 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 | ST003424 |
| Study Title | Analysis of lipid composition of HeLa and U2OS CLN8 KO cultured cells |
| Study Summary | This study was conducted to explore whether other members of the human TLCD protein family, specifically CLN8 (also known as TLCD6), function as lysophospholipid acyltransferases, similar to TLCD1. The CLN8 gene was selected for investigation due to its association with Batten disease, a severe neurodegenerative lysosomal storage disorder in children. CLN8 is known to localize to the ER-Golgi network and has been previously identified as a cargo receptor for lysosomal proteins. To investigate its potential role in phospholipid metabolism, CRISPR-edited CLN8 knockout (KO) HeLa and U-2OS cell pools were generated, and their lipid profiles were compared to wild-type controls using untargeted lipidomics. The study aimed to determine whether CLN8 plays a role in the phospholipid metabolism, expanding the understanding of the functions of TLCD family proteins in lipid metabolism. This study is a part of a manuscript TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid composition (currently under review at Science Advances). |
| Institute | University of Cambridge |
| Last Name | Petkevicius |
| First Name | Kasparas |
| Address | The Keith Peters Building |
| kp416@mrc-mbu.cam.ac.uk | |
| Phone | 07500233355 |
| Submit Date | 2024-08-20 |
| Publications | TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid composition (currently under review at Science Advances) |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-11-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002115 |
| Project DOI: | doi: 10.21228/M87C0V |
| Project Title: | TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid composition |
| Project Summary: | The diversity of cellular phospholipids, crucial for membrane homeostasis and function, arises from enzymatic remodeling of their fatty acyl chains. In this work, we reveal that poorly understood TRAM–LAG1–CLN8 domain-containing (TLCD) proteins are phospholipid remodeling enzymes. We demonstrate that TLCD1 is an evolutionarily conserved lysophosphatidylethanolamine acyltransferase, which regulates cellular phospholipid composition and generates novel fatty acid and thiamine (vitamin B1) esters as its secondary products. Furthermore, we establish that human TLCD protein CLN8, mutations in which cause fatal neurodegenerative Batten disease, is a lysophosphatidylglycerol acyltransferase. We show that CLN8 catalyzes the essential step in the biosynthesis of bis(monoacylglycero)phosphate, a phospholipid critical for lysosome function. Our study unveils a new family of acyltransferases integral to cellular membrane phospholipid homeostasis and human disease. |
| Institute: | University of Cambridge |
| Last Name: | Petkevicius |
| First Name: | Kasparas |
| Address: | The Keith Peters Building, Cambridge, Cambridgeshire, CB2 0XY, United Kingdom |
| Email: | kp416@mrc-mbu.cam.ac.uk |
| Phone: | +447500233355 |
| Publications: | TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid composition |
Subject:
| Subject ID: | SU003551 |
| 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 | Genotype | Sample source | Transfection | Replicate |
|---|---|---|---|---|---|
| SA377656 | HeLa CLN8 KO pool + CLN8-HA Replicate 1 | CLN8 KO | HeLa | CLN8-HA | 1 |
| SA377657 | HeLa CLN8 KO pool + CLN8-HA Replicate 2 | CLN8 KO | HeLa | CLN8-HA | 2 |
| SA377658 | HeLa CLN8 KO pool + CLN8-HA Replicate 3 | CLN8 KO | HeLa | CLN8-HA | 3 |
| SA377659 | HeLa CLN8 KO pool + EV Replicate 1 | CLN8 KO | HeLa | Empty vector | 1 |
| SA377660 | HeLa CLN8 KO pool + EV Replicate 2 | CLN8 KO | HeLa | Empty vector | 2 |
| SA377661 | HeLa CLN8 KO pool + EV Replicate 3 | CLN8 KO | HeLa | Empty vector | 3 |
| SA377662 | HeLa CLN8 KO poolReplicate 1 | CLN8 KO | HeLa | None | 1 |
| SA377663 | HeLa CLN8 KO poolReplicate 2 | CLN8 KO | HeLa | None | 2 |
| SA377664 | HeLa CLN8 KO poolReplicate 3 | CLN8 KO | HeLa | None | 3 |
| SA377665 | U-2OS CLN8 KO pool + CLN8-HA Replicate 1 | CLN8 KO | U2OS | CLN8-HA | 1 |
| SA377666 | U-2OS CLN8 KO pool + CLN8-HA Replicate 2 | CLN8 KO | U2OS | CLN8-HA | 2 |
| SA377667 | U-2OS CLN8 KO pool + CLN8-HA Replicate 3 | CLN8 KO | U2OS | CLN8-HA | 3 |
| SA377668 | U-2OS CLN8 KO pool + EV Replicate 1 | CLN8 KO | U2OS | Empty vector | 1 |
| SA377669 | U-2OS CLN8 KO pool + EV Replicate 2 | CLN8 KO | U2OS | Empty vector | 2 |
| SA377670 | U-2OS CLN8 KO pool + EV Replicate 3 | CLN8 KO | U2OS | Empty vector | 3 |
| SA377671 | U-2OS CLN8 KO poolReplicate 1 | CLN8 KO | U2OS | None | 1 |
| SA377672 | U-2OS CLN8 KO poolReplicate 2 | CLN8 KO | U2OS | None | 2 |
| SA377673 | U-2OS CLN8 KO poolReplicate 3 | CLN8 KO | U2OS | None | 3 |
| SA377674 | Cell process blank | Process blank | Process blank | Process blank | Process blank |
| SA377675 | HeLa Wild-Type poolReplicate 1 | Wild-type | HeLa | None | 1 |
| SA377676 | HeLa Wild-Type poolReplicate 2 | Wild-type | HeLa | None | 2 |
| SA377677 | HeLa Wild-Type poolReplicate 3 | Wild-type | HeLa | None | 3 |
| SA377678 | U-2OS Wild-Type poolReplicate 1 | Wild-type | U2OS | None | 1 |
| SA377679 | U-2OS Wild-Type poolReplicate 2 | Wild-type | U2OS | None | 2 |
| SA377680 | U-2OS Wild-Type poolReplicate 3 | Wild-type | U2OS | None | 3 |
| Showing results 1 to 25 of 25 |
Collection:
| Collection ID: | CO003544 |
| Collection Summary: | All cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM, 11965084, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS, Sigma) at 37°C and 5% CO2. To collect samples for lipid measurements, confluent cells in 6-well plates were swiftly washed twice with 3 mL of ice-cold PBS on ice, then scraped into 500 μl of ice-cold PBS on ice using inverted 200 μl pipette tip. The resulting cell suspension was transferred into pre-chilled 2 mL plastic vials used for lipid extraction (3469-11, Thermo Scientific), and centrifuged at 500 g, 4°C for 5 min. The supernatant was aspirated and the cell pellet was snap-frozen and stored at -70°C. One well of a 6-well plate yielded one replicate in analysis. |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003560 |
| Treatment Summary: | Cells were either untreated (collected under standard culturing conditions) or transfected using FuGENE® HD Transfection Reagent (E2311, Promega) according to the manufacturer’s protocol. For a single well of a 6-well plate, 20 μl of Opti-MEM (31985062, Thermo Fisher), 0.4 μg plasmid DNA and 1.2 μl of FuGENE HD was used. Medium was refreshed the next day and cells were analyzed 48-72 hours post transfection. |
Sample Preparation:
| Sampleprep ID: | SP003558 |
| Sampleprep Summary: | The extraction of total lipids from cellular matrices, immunoprecipitated mitochondria and in vitro assays was conducted employing the butanol-methanol (BUME) method, as described in detail (37). Note that the chloroform-methanol lipid extraction methods result in the partitioning of acyl-thiamines into the polar phase. 2 mL screw cap plastic tubes were used (3469-11, Thermo Scientific), and a blank extraction was always performed in parallel to account for the plastic-related contaminants. The extraction commenced with the homogenization of frozen cell pellets, mitochondrial beads or in vitro assay mixtures in a 0.5 mL ice-cold solution of butanol to methanol in a 3:1 ratio. For lipidomics samples, BUME solution was enriched with SPLASH internal standard mix (330707, Avanti Polar Lipids). For the extraction, a further 0.5 mL of 1% acetic acid and 0.5 mL of a heptane:ethyl acetate 3:1 mixture were added, followed by vigorous vortexing for a total of 5 minutes. The mixture was then centrifuged at 6000 g for 5 minutes, allowing for the separation of phases, after which the upper organic phase was carefully decanted into glass vials. A second extraction was conducted on the remaining aqueous phase, with the newly acquired upper phase being combined in the same glass vials as the first. Post extraction, the solvents were evaporated under a stream of nitrogen, and the resultant dry lipid extracts were preserved at −70°C pending further analysis. |
Combined analysis:
| Analysis ID | AN005623 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Shimadzu 10A |
| Column | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | nmol |
Chromatography:
| Chromatography ID: | CH004273 |
| Chromatography Summary: | Dried samples were reconstituted in 100 μL of isopropanol, acetonitrile, and water (2:1:1 ratio) and thoroughly vortexed. Liquid chromatography was conducted using a Shimadzu HPLC System, with 10 μL of the sample introduced onto a Waters Acquity Premier UPLC® CSH column (1.7 μm pore size, 2.1 mm × 50 mm), which was maintained at 55°C. The mobile phase A comprised a 6:4 ratio of acetonitrile to water with 10 mM ammonium formate, and mobile phase B consisted of a 9:1 ratio of isopropanol to acetonitrile with 10 mM ammonium formate. A flow rate of 500 μL per minute was maintained with a gradient protocol for mobile phase B as follows: 0.00 minutes_40% mobile phase B; 1.5 minutes_40% mobile phase B; 8.00 minutes_99% mobile phase B; 10.00 minutes_99% mobile phase B; 10.10 minutes_40% mobile phase B; 12.00 minutes_40% mobile phase. The sample injection needle was rinsed with a 9:1 isopropanol and acetonitrile solution (strong wash) and isopropanol, acetonitrile, and water (2:1:1, weak wash). |
| Instrument Name: | Shimadzu 10A |
| Column Name: | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 55°C |
| Flow Gradient: | Gradient protocol for mobile phase B as follows: 0.00 minutes_40% mobile phase B; 1.5 minutes_40% mobile phase B; 8.00 minutes_99% mobile phase B; 10.00 minutes_99% mobile phase B; 10.10 minutes_40% mobile phase B; 12.00 minutes_40% mobile phase. |
| Flow Rate: | 500 μL/min |
| Solvent A: | 60% Acetonitrile/40% Water; 10 mM Ammonium formate |
| Solvent B: | 90% Isopropanol/10% Acetonitrile; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005347 |
| Analysis ID: | AN005623 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometric detection was carried out on a ThermoFisher Scientific Q-Exactive Orbitrap equipped with a heated electrospray ionization source. The mass spectrometer was calibrated immediately before sample analysis using positive and negative ionization calibration solution (recommended by Thermo Scientific). The electrospray ionization parameters were optimized with a 50:50 mix of mobile phase A and B for spray stability, setting the capillary temperature at 300 °C, source heater temperature at 420 °C, with the sheath, auxiliary, and spare gas flows at specific arbitrary units (40, 15 and 3, respectively), and source voltage at 4 kV. The mass spectrometer operated at a scan rate of 4 Hz, yielding a resolution of 35,000 at m/z 200, over a full-scan range from m/z 120 to 1800, with continuous switching between positive and negative modes. |
| Ion Mode: | UNSPECIFIED |
