Summary of Study ST003418
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 | ST003418 |
| Study Title | Analysis of lipid composition of TLCD1 KO cultured cells |
| Study Summary | This study was conducted to explore the biochemical function of the TLCD1 protein and its role in regulating cellular membrane lipid composition. Phosphatidylethanolamine (PE), a key membrane phospholipid, is particularly abundant in mitochondria. Previous research suggested that TLCD1, along with its homologs, might influence PE composition and be involved in the progression of non-alcoholic fatty liver disease. However, the specific mechanisms by which TLCD1 regulates lipid composition were not well understood. To address this gap, TLCD1 knockout (KO) HeLa and U2OS human cell models were engineered and subjected to untargeted lipidomics analysis, aiming to shed light on the role of TLCD1 in cellular phospholipid metabolism. This study is part of the manuscript (Sheokand et al), 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 | 2024-11-11 |
| 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: | SU003545 |
| 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 | Clonality | Replicate |
|---|---|---|---|---|---|
| SA377481 | Cell process blank | Process blank | Process blank | Process blank | Process blank |
| SA377482 | HeLa TLCD1 KO poolReplicate 1 | TLCD1 KO | HeLa | Cell pool | 1 |
| SA377483 | HeLa TLCD1 KO poolReplicate 2 | TLCD1 KO | HeLa | Cell pool | 2 |
| SA377484 | HeLa TLCD1 KO poolReplicate 3 | TLCD1 KO | HeLa | Cell pool | 3 |
| SA377485 | HeLa TLCD1 KO cloneReplicate 1 | TLCD1 KO | HeLa | Clone derived | 1 |
| SA377486 | HeLa TLCD1 KO cloneReplicate 2 | TLCD1 KO | HeLa | Clone derived | 2 |
| SA377487 | HeLa TLCD1 KO cloneReplicate 3 | TLCD1 KO | HeLa | Clone derived | 3 |
| SA377488 | U20S TLCD1 KO poolReplicate 1 | TLCD1 KO | U2OS | Cell pool | 1 |
| SA377489 | U20S TLCD1 KO poolReplicate 2 | TLCD1 KO | U2OS | Cell pool | 2 |
| SA377490 | U20S TLCD1 KO poolReplicate 3 | TLCD1 KO | U2OS | Cell pool | 3 |
| SA377491 | U20S TLCD1 KO cloneReplicate 1 | TLCD1 KO | U2OS | Clone derived | 1 |
| SA377492 | U20S TLCD1 KO cloneReplicate 2 | TLCD1 KO | U2OS | Clone derived | 2 |
| SA377493 | U20S TLCD1 KO cloneReplicate 3 | TLCD1 KO | U2OS | Clone derived | 3 |
| SA377494 | HeLa Wild-Type poolReplicate 1 | Wild-type | HeLa | Cell pool | 1 |
| SA377495 | HeLa Wild-Type poolReplicate 2 | Wild-type | HeLa | Cell pool | 2 |
| SA377496 | HeLa WildType poolReplicate 3 | Wild-type | HeLa | Cell pool | 3 |
| SA377497 | HeLa Wild-Type clone 2Replicate 1 | Wild-type | HeLa | Clone derived | 1 |
| SA377498 | HeLa WildType clone 1Replicate 1 | Wild-type | HeLa | Clone derived | 1 |
| SA377499 | HeLa WildType clone 2Replicate 2 | Wild-type | HeLa | Clone derived | 2 |
| SA377500 | HeLa Wild-Type clone 1Replicate 2 | Wild-type | HeLa | Clone derived | 2 |
| SA377501 | HeLa Wild-Type clone 1Replicate 3 | Wild-type | HeLa | Clone derived | 3 |
| SA377502 | HeLa Wild-Type clone 2Replicate 3 | Wild-type | HeLa | Clone derived | 3 |
| SA377503 | U20S WildType poolReplicate 1 | Wild-type | U2OS | Cell pool | 1 |
| SA377504 | U20S Wild-Type poolReplicate 2 | Wild-type | U2OS | Cell pool | 2 |
| SA377505 | U20S Wild-Type poolReplicate 3 | Wild-type | U2OS | Cell pool | 3 |
| SA377506 | U20S Wild-Type clone 2Replicate 1 | Wild-type | U2OS | Clone derived | 1 |
| SA377507 | U20S Wild-Type clone 1Replicate 1 | Wild-type | U2OS | Clone derived | 1 |
| SA377508 | U20S Wild-Type clone 2Replicate 2 | Wild-type | U2OS | Clone derived | 2 |
| SA377509 | U20S Wild-Type clone 1Replicate 2 | Wild-type | U2OS | Clone derived | 2 |
| SA377510 | U20S Wild-Type clone 1Replicate 3 | Wild-type | U2OS | Clone derived | 3 |
| SA377511 | U20S Wild-Type clone 2Replicate 3 | Wild-type | U2OS | Clone derived | 3 |
| Showing results 1 to 31 of 31 |
Collection:
| Collection ID: | CO003538 |
| 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: | TR003554 |
| Treatment Summary: | Cells were untreated (collected under standard culturing conditions). |
Sample Preparation:
| Sampleprep ID: | SP003552 |
| 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 | AN005617 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| 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: | CH004267 |
| 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: | Waters Acquity |
| 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: | MS005341 |
| Analysis ID: | AN005617 |
| 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 |
