Summary of Study ST003732
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 PR002318. The data can be accessed directly via it's Project DOI: 10.21228/M80R8H This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST003732 |
| Study Title | Lipidomic assessment of sphingomyelinase, cholesterol oxidase and cyclodextrin effect in Jurkat Paris |
| Study Summary | Metabolomics and its branch lipidomics are the youngest disciplines in the group of ‘omics’ sciences and both have drawn increasing attention in the clinical arena. This interest is premised on the close association between the metabolites and the phenotype, which posits metabolomics as a powerful tool for the real-time assessment of a given metabolic state. Lipids are the fundamental building blocks of biological membranes. Aside of this structural role, lipids also play multiple important functions in biological systems as energy storage entities and participating in metabolic regulation, membrane trafficking, signaling, proliferation or apoptosis. Due to this diverse range of functions and together with the noteworthy technological improvements and the depth of basic research over the past decade, the field of lipidomics has established itself as a key player in the creation of specific biomarkers for disease diagnosis and for the purpose of drug therapy monitoring. Endogenous metabolic profiles of cell samples coming from Jurkat Paris, have been studied using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Mass spectrometry coupled to ultra-high performance liquid chromatography is well suited to such analyses due to its high sensitivity, large coverage over different classes of metabolites, high throughput capacity, and wide dynamic range. The general aim of the project was to evaluate potential lipidomic differences between three different treatments applied to these two human cell types and determine possible phenotypical biomarkers by statistical data analysis. A total of 16 cell samples from Jurkat Paris (JKP), were received from the National Centre for Biotechnology (Madrid, Spain) and classified according to the treatment received: sphingomyelinase (SMase), cholesterol oxidase (CoAse), cyclodextrin (CD), and non-treated (Control). The aim of the study is to analyze the effect of cholesterol depletion on the function of the chemokine receptor CXCR4. Methyl beta-cyclodextrin (MCD) has been widely used to investigate the role of cholesterol in GPCR biology. However, this is a very aggressive treatment, as cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. We thus decided to use ChOx to promote a weak modulation of cholesterol levels at the cell membrane. This enzyme allows a specific oxidation of raft cholesterol into cholestenone that does not inhibit the activation of either Jurkat cells or T CD4+ lymphocytes. The lipidomic analysis of cells treated with ChOx using UHPLC-ESI-MS, demonstrated no effects on sphingomyelins and ceramides expression and a very moderate decrease in cholesterol levels. As control, cells treated with bSMase showed a complete breakdown of sphingomyelins with no effects on cholesterol levels and MCD promoted a severe reduction in cholesterol levels compatible with the strong defects observed in the conformations adopted by CXCR4. |
| Institute | Centro Nacional De Biotecnologia |
| Last Name | Mellado |
| First Name | Mario |
| Address | C/ Darwin 3, 28049, Madrid, Spain |
| bsoler@cnb.csic.es | |
| Phone | +34 915854660 |
| Submit Date | 2025-01-22 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2026-01-22 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002318 |
| Project DOI: | doi: 10.21228/M80R8H |
| Project Title: | A moderate depletion of cholesterol inhibits CXCL12 mediated direct cell migration in human T cells |
| Project Summary: | Cholesterol is a major component of mammalian cell membranes. It modulates the lipid bilayer properties and also affects the conformations adopted by membrane receptors. Here, we show that moderate cholesterol depletion achieved through the use of cholesterol oxidase (ChOx) increased membrane fluidity and hindered the capacity of T cells to move towards CXCL12 gradients. While fluorescence resonance energy transfer (FRET) studies revealed changes in the conformation of CXCR4, no significant alterations in ligand binding, in other chemokine-mediated signaling pathways, or even in the dynamics of the receptors at the cell membrane were observed. Furthermore, immunocytochemistry approaches indicated that ChOx treatment did not affect the integrity of lipid enriched microdomains containing CXCR4, thereby preserving ligand-mediated receptor nanoclustering. In addition, under these experimental conditions, we detected higher CXCL12-mediated beta-1 integrins activation and increased cell adhesion to fibronectin. Taken together, these findings suggest that by targeting the cell membrane, where these receptors are embedded, it is possible to modulate certain chemokine-mediated functions. |
| Institute: | Centro Nacional De Biotecnologia |
| Last Name: | Mellado |
| First Name: | Mario |
| Address: | C/ Darwin 3, 28049, Madrid, Spain |
| Email: | mmellado@cnb.csic.es |
| Phone: | 915854660 |
Subject:
| Subject ID: | SU003864 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA407440 | JKP CD 3 | CD |
| SA407441 | JKP CD 4 | CD |
| SA407442 | JKP CD 2 | CD |
| SA407443 | JKP CD 1 | CD |
| SA407445 | JKP cell blank | Cell blank |
| SA407444 | JKP cell sample | Cell Sample |
| SA407446 | JKP CoAse 3 | CoAsa |
| SA407447 | JKP CoAse 1 | CoAsa |
| SA407448 | JKP CoAse 4 | CoAsa |
| SA407449 | JKP CoAse 2 | CoAsa |
| SA407450 | JKP Ctrl 2 | Ctrl |
| SA407451 | JKP Ctrl 4 | Ctrl |
| SA407452 | JKP Ctrl 3 | Ctrl |
| SA407453 | JKP Ctrl 1 | Ctrl |
| SA407454 | JKP Smase 2 | Smasa |
| SA407455 | JKP Smase 1 | Smasa |
| SA407456 | JKP Smase 4 | Smasa |
| SA407457 | JKP Smase 3 | Smasa |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO003857 |
| Collection Summary: | A total of 16 cell samples from Jurkat Paris (JKP), were received from the National Centre for Biotechnology (Madrid, Spain). |
| Sample Type: | Jurkat T-cells |
Treatment:
| Treatment ID: | TR003873 |
| Treatment Summary: | 16 cell samples derived from Jurkat (JK) cells. The samples were subjected to four distinct treatments: bacterial Sphingomyelinase (bSMase, 0.5 U/mL, 3h, 37ºC), Cholesterol Oxidase (ChOx, 5 U/mL, 2h, 37ºC), Methyl-beta-Cyclodextrin (MCD, 5mM, 30 min, 37ºC) or Control (untreated) prior to lipid extraction. |
Sample Preparation:
| Sampleprep ID: | SP003870 |
| Sampleprep Summary: | Lipid extraction was accomplished through a fractionation process into pools of lipids with similar physicochemical properties, using appropriate combinations of organic solvents. A chloroform/methanol extraction was employed for isolation of apolar lipids and, then, an UHPLC-MS platform was used for the optimal profiling of glycerolipids, cholesterol esters, sphingolipids and glycerophospholipids. LC-MS-grade solvents were purchased from Sigma Aldrich (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA). Reference metabolite standard compounds were obtained from Sigma Aldrich, Avanti Polar Lipids (Alabaster, AL), and Larodan Fine Chemicals (Malmö, Sweden). Cells (7.5 x 10^6) were resuspended in cold water and briefly mixed. Proteins were precipitated from the lysed cell samples by adding a cold solution of chloroform/methanol. Samples were incubated for 30 minutes at -20 °C and then vortexed. Then, different fractions of these samples were obtained and the following procedures were applied to extract the target analytes’ chemical class: glycerolipids, cholesteryl esters, sphingolipids and glycerophospholipids profiling. Cold water was added to a fraction of 500 μL of the sample. Then the sample was incubated for additional 30 minutes at -20 °C. After that, samples were vortex-mixed and centrifuged at 18,000 x g for 15 minutes at 4 ºC to facilitate the separation of the organic and aqueous phases. The organic layer was collected, dried under vacuum and resuspended in acetonitrile/isopropanol (1:1). Then, samples were transferred to vials for UHPLC-MS analysis. |
Combined analysis:
| Analysis ID | AN006123 |
|---|---|
| Chromatography ID | CH004650 |
| MS ID | MS005829 |
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Waters Xevo G2 QTof |
| Ion Mode | POSITIVE |
| Units | ppm |
Chromatography:
| Chromatography ID: | CH004650 |
| Chromatography Summary: | Chromatography was performed using ACQUITY UPLC system (Waters Corp., Milford, USA), coupled to a Xevo G2 QTof (Waters Corp.) mass spectrometer. Randomized sample injections were performed, with calibration and validation extracts uniformly interspersed throughout the entire batch run. A linear gradient was used at a flow rate of 0.4 mL/min, with solvent A: H2O:Acetonitrile (2:3) 10 mM ammonium formate and solvent B Acetonitrile: isopropanol (1:9) 10 mM ammonium formate. The gradient starts at 40% B, increasing to 99.9% in 10 min. This composition was then maintained for 5 min for column cleaning. Finally, the column was conditioned for a further 2 min with the starting conditions. |
| Methods Filename: | LC-MS_analysis.txt |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 60 °C |
| Flow Gradient: | 40% B, 0 min; 100% B, 10 min; 100% B, 15 min; 40% B, 17 min |
| Flow Rate: | 0.4 mL/min |
| Solvent A: | 40% water/60% acetonitrile; 10 mM Ammonium formate |
| Solvent B: | 10% Acetonitrile/90% isopropanol; 10 mM Ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005829 |
| Analysis ID: | AN006123 |
| Instrument Name: | Waters Xevo G2 QTof |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | For monoacylglycerides (MAG), diacylglycerides (DAG), triacylglycerides (TAG), cholesteryl esters (ChoE), glycerophosphocholines (PC), glycerophosphoethanolamines (PE), glycerophosphoinositols (PI), sphingomyelins (SM), ceramides (Cer), and monohexosylceramides (CMH) species, a theoretical m/z database was first generated for all possible combinations of fatty acid derived moieties. The association of detected Rt-m/z pairs with lipid species contained in the theoretical database was subsequently established either by comparison of their accurate mass spectra and chromatographic retention times with those obtained using available reference standards or, where these were not available, by accurate mass MS/MS fragment ion analysis. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | LC-MS_analysis.txt |
