Summary of Study ST003420

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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003420
Study TitleAnalysis of lipid composition of control, YPR114w and YJR116w yeast mutants grown under exponential phase
Study SummaryThis study was conducted to investigate whether the enzymatic activity of TLCD1, a protein implicated in membrane phospholipid regulation, is conserved in yeast. The research aimed to explore the functions of the TLCD1 homologs, YPR114w and YJR116w, in S. cerevisiae. Deletion mutants of these genes were generated to assess their role in lipid metabolism, particularly in relation to phosphatidylethanolamine (PE) composition. By performing untargeted lipidomics analysis of mentioned yeast strains cultured under exponential growth phase, the study sought to determine if YPR114w and YJR116w contribute to lipid regulation in a manner similar to TLCD1 in higher organisms. This work is part of the manuscript TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid composition (currently under review at Science Advances).
Institute
University of Cambridge
Last NamePetkevicius
First NameKasparas
AddressThe Keith Peters Building
Emailkp416@mrc-mbu.cam.ac.uk
Phone07500233355
Submit Date2024-08-20
PublicationsTRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid composition (currently under review at Science Advances)
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-11-11
Release Version1
Kasparas Petkevicius Kasparas Petkevicius
https://dx.doi.org/10.21228/M87C0V
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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:SU003547
Subject Type:Cultured cells
Subject Species:Saccharomyces cerevisiae
Taxonomy ID:4932

Factors:

Subject type: Cultured cells; Subject species: Saccharomyces cerevisiae (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Sample source Replicate
SA377549Yeast process blankProcess blank Process blank Process blank
SA377550Wild-type replicate 1Wild-type Saccharomyces cerevisiae 1
SA377551Wild-type replicate 2Wild-type Saccharomyces cerevisiae 2
SA377552Wild-type replicate 3Wild-type Saccharomyces cerevisiae 3
SA377553YJR116w KO replicate 1YJR116w KO Saccharomyces cerevisiae 1
SA377554YJR116w KO replicate 2YJR116w KO Saccharomyces cerevisiae 2
SA377555YJR116w KO replicate 3YJR116w KO Saccharomyces cerevisiae 3
SA377556YPR114w KO replicate 1YPR114w KO Saccharomyces cerevisiae 1
SA377557YPR114w KO replicate 2YPR114w KO Saccharomyces cerevisiae 2
SA377558YPR114w KO replicate 3YPR114w KO Saccharomyces cerevisiae 3
Showing results 1 to 10 of 10

Collection:

Collection ID:CO003540
Collection Summary:Yeast cells were grown in synthetic medium (SC) containing 2% glucose, 0.17% yeast nitrogen base (Cat. No. 233520, Difco, BD, Franklin Lakes, NJ), 0.5% ammonium sulfate, and amino acid drop-out (60 mg/L leucine, 55 mg/L adenine, 55 mg/L uracil, 55 mg/L tyrosine, 20 mg/L of arginine, 10 mg/L histidine, 60 mg/L isoleucine, 40 mg/L lysine, 60 mg/L phenylalanine, 50 mg/L threonine, 10 mg/L methionine, 40 mg/L tryptophan). For lipidomics, cells were grown overnight to exponential phase (OD600 0.4-0.6), then were washed once in water and snap frozen in liquid nitrogen. For acyl-thiamine measurements, cultures were grown to the exponential phase overnight, then split in two; one half was left untreated while the second half was supplemented with 100 mM thiamine. All cultures were then grown for 2h 30 min. Cells were washed once in water and snap frozen in liquid nitrogen.
Sample Type:Yeast cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003556
Treatment Summary:Cells were untreated (collected under standard culturing conditions).

Sample Preparation:

Sampleprep ID:SP003554
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 AN005619
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:CH004269
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:990% Isopropanol/10% Acetonitrile; 10 mM ammonium formate
Chromatography Type:Reversed phase

MS:

MS ID:MS005343
Analysis ID:AN005619
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
  logo