Summary of Study ST003419

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	ST003419
Study Title	Analysis of lipid composition of the mitochondria isolated from TLCD1 KO cell models
Study Summary	This study was conducted to explore the biochemical function of the TLCD1 protein and its role in regulating mitochondrial 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 with genetically tagged mitochondria were engineered. Mitochondria from these cell lines were isolated by immunoprecipitation and subjected to untargeted lipidomics analysis, aiming to shed light on the role of TLCD1 in mitochondrial 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, Cambridge, Cambridgeshire, CB2 0XY, United Kingdom
Email	kp416@mrc-mbu.cam.ac.uk
Phone	+447500233355
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:	SU003546
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	MitoTag	Sample source	Replicate
SA377512	Cell process blank	Process blank	Process blank	Process blank	Process blank
SA377513	MitoIP HeLa TLCD1 KO pool HA R1	TLCD1 KO	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	1
SA377514	MitoIP HeLa TLCD1 KO pool HA R2	TLCD1 KO	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	2
SA377515	MitoIP HeLa TLCD1 KO pool HA R3	TLCD1 KO	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	3
SA377516	MitoIP HeLa TLCD1 KO pool HA R4	TLCD1 KO	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	4
SA377517	MitoIP HeLa TLCD1 KO pool HA R5	TLCD1 KO	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	5
SA377518	MitoIP HeLa TLCD1 KO pool HA R6	TLCD1 KO	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	6
SA377519	WholeCell HeLa TLCD1 KO pool HA R1	TLCD1 KO	3xHA-EGFP-OMP25	Whole cell lipidomics	1
SA377520	WholeCell HeLa TLCD1 KO pool HA R2	TLCD1 KO	3xHA-EGFP-OMP25	Whole cell lipidomics	2
SA377521	WholeCell HeLa TLCD1 KO pool HA R3	TLCD1 KO	3xHA-EGFP-OMP25	Whole cell lipidomics	3
SA377522	MitoIP HeLa TLCD1 KO pool MYC R1	TLCD1 KO	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	1
SA377523	MitoIP HeLa TLCD1 KO pool MYC R2	TLCD1 KO	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	2
SA377524	MitoIP HeLa TLCD1 KO pool MYC R3	TLCD1 KO	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	3
SA377525	MitoIP HeLa TLCD1 KO pool MYC R4	TLCD1 KO	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	4
SA377526	MitoIP HeLa TLCD1 KO pool MYC R5	TLCD1 KO	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	5
SA377527	MitoIP HeLa TLCD1 KO pool MYC R6	TLCD1 KO	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	6
SA377528	WholeCell HeLa TLCD1 KO pool MYC R1	TLCD1 KO	3xMYC-EGFP-OMP25	Whole cell lipidomics	1
SA377529	WholeCell HeLa TLCD1 KO pool MYC R2	TLCD1 KO	3xMYC-EGFP-OMP25	Whole cell lipidomics	2
SA377530	WholeCell HeLa TLCD1 KO pool MYC R3	TLCD1 KO	3xMYC-EGFP-OMP25	Whole cell lipidomics	3
SA377531	MitoIP HeLa Wild-Type pool HA R1	Wild-type	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	1
SA377532	MitoIP HeLa Wild-Type pool HA R2	Wild-type	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	2
SA377533	MitoIP HeLa Wild-Type pool HA R3	Wild-type	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	3
SA377534	MitoIP HeLa Wild-Type pool HA R4	Wild-type	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	4
SA377535	MitoIP HeLa Wild-Type pool HA R5	Wild-type	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	5
SA377536	MitoIP HeLa Wild-Type pool HA R6	Wild-type	3xHA-EGFP-OMP25	Mitochondria IP lipidomics	6
SA377537	WholeCell HeLa Wild-Type pool HA R1	Wild-type	3xHA-EGFP-OMP25	Whole cell lipidomics	1
SA377538	WholeCell HeLa Wild-Type pool HA R2	Wild-type	3xHA-EGFP-OMP25	Whole cell lipidomics	2
SA377539	WholeCell HeLa Wild-Type pool HA R3	Wild-type	3xHA-EGFP-OMP25	Whole cell lipidomics	3
SA377540	MitoIP HeLa Wild-Type pool MYC R1	Wild-type	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	1
SA377541	MitoIP HeLa Wild-Type pool MYC R2	Wild-type	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	2
SA377542	MitoIP HeLa Wild-Type pool MYC R3	Wild-type	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	3
SA377543	MitoIP HeLa Wild-Type pool MYC R4	Wild-type	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	4
SA377544	MitoIP HeLa Wild-Type pool MYC R5	Wild-type	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	5
SA377545	MitoIP HeLa Wild-Type pool MYC R6	Wild-type	3xMYC-EGFP-OMP25	Mitochondria IP lipidomics	6
SA377546	WholeCell HeLa Wild-Type pool MYC R1	Wild-type	3xMYC-EGFP-OMP25	Whole cell lipidomics	1
SA377547	WholeCell HeLa Wild-Type pool MYC R2	Wild-type	3xMYC-EGFP-OMP25	Whole cell lipidomics	2
SA377548	WholeCell HeLa Wild-Type pool MYC R3	Wild-type	3xMYC-EGFP-OMP25	Whole cell lipidomics	3

Showing results 1 to 37 of 37

Showing results 1 to 37 of 37

Collection:

Collection ID:	CO003539
Collection Summary:	All the mito-IP steps were conducted using ice-cold buffers either in a cold-room or on ice and all centrifugation steps were carried out at 4°C. Cells were washed twice with 30 mL of PBS and then harvested in 1 mL of mito-IP buffer (10 mM KH2PO4, 137 mM KCl). Cells were collected at 700 x g for 5 min and resuspended in 1 mL of mito-IP buffer containing protease inhibitors (Thermo Fisher Scientific, 78429) per 15 cm plate and then lysed using 25 passes through a Dounce homogenizer. After 1,500 x g centrifugation of the lysate for 10 min, a post-nuclear supernatant (PNS) was obtained and incubated with 100 μL of anti-HA magnetic beads (Sigma-Aldrich, SAE0197), previously equilibrated in the mito-IP buffer. Finally, beads were collected using a magnetic rack, washed three times with 1 mL of mito-IP buffer for 5 min each, and dried beads were subsequently frozen in 2 mL glass autosampler vials.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003555
Treatment Summary:	No treatment, cells were collected under normal growth conditions and immunoprecipitation was performed as described under 'collection.

Sample Preparation:

Sampleprep ID:	SP003553
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.

Sampleprep ID:

SP003553

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	AN005618
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:	CH004268
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:	MS005342
Analysis ID:	AN005618
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