Summary of Study ST002150

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 PR001363. The data can be accessed directly via it's Project DOI: 10.21228/M8J708 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	ST002150
Study Title	Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed cell migration in human T cells
Study Summary	Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-molecule dynamic approaches to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with neutral sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration
Institute	Universidad CEU San Pablo
Last Name	Gonzalez-Riano
First Name	Carolina
Address	km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501
Email	carolina.gonzalezriano@ceu.es
Phone	646251045
Submit Date	2022-04-22
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2022-05-09
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001363
Project DOI:	doi: 10.21228/M8J708
Project Title:	Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed cell migration in human T cells
Project Summary:	Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-molecule dynamic approaches to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with neutral sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.
Institute:	Universidad CEU San Pablo
Department:	Center of Metabolomics and Bioanalysis
Last Name:	Gonzalez-Riano
First Name:	Carolina
Address:	km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501
Email:	carolina.gonzalezriano@ceu.es
Phone:	646251045

Subject:

Subject ID:	SU002236
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	Factor1
SA205954	Blasto_SMasa_9	CASE
SA205955	Blasto_SMasa_3	CASE
SA205956	Blasto_SMasa_2	CASE
SA205957	Jurkat_SMasa_1	CASE
SA205958	Jurkat_SMasa_3	CASE
SA205959	Jurkat_SMasa_7	CASE
SA205960	Jurkat_SMasa_4	CASE
SA205961	Blasto_SMasa_10	CASE
SA205962	Jurkat_SMase_10	CASE
SA205963	Blasto_SMasa_6	CASE
SA205964	Blasto_Control_8	CONTROL
SA205965	Blasto_Control_7	CONTROL
SA205966	Jurkat_Control_9	CONTROL
SA205967	Blasto_Control_4	CONTROL
SA205968	Jurkat_Control_2	CONTROL
SA205969	Blasto_Control_5	CONTROL
SA205970	Jurkat_Control_5	CONTROL
SA205971	Blasto_Control_1	CONTROL
SA205972	Jurkat_Control_8	CONTROL
SA205973	Jurkat_Control_6	CONTROL
SA205974	QC_2_J	QC
SA205975	QC_3_J	QC
SA205976	QC_1_J_2	QC
SA205977	QC_3_B	QC
SA205978	QC_1_B	QC
SA205979	QC_2_B	QC
SA205980	QC_1_J_1	QC

Showing results 1 to 27 of 27

Showing results 1 to 27 of 27

Collection:

Collection ID:	CO002229
Collection Summary:	HEK-293T cells were obtained from the ATCC (CRL-11268) and human Jurkat leukemia CD4+ cells were kindly donated by Dr. J. Alcamí (Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain). When needed, Jurkat cells lacking endogenous CXCR4 expression (Jurkat-/-) were transiently transfected with CXCR4-AcGFP (20 µg; JK-/-X4) using a BioRad electroporator (20 × 106 cells/400 µL RPMI 1640 with 10% fetal calf serum) and analyzed 24 hours later. Human peripheral blood mononuclear cells were isolated from buffy coats by centrifugation through FicollPaque PLUS density gradients (GE Healthcare, Wakuesha, WI) at 760 × g for 30 minutes at room temperature (RT). They were then in vitro activated with 20 U/mL of IL-2 (Teceleukin; Roche, Nutley, NJ) and 5 µg/mL phytohemagglutinin PHA (Roche) to generate T cell blasts.
Sample Type:	HEK cells

Treatment:

Treatment ID:	TR002248
Treatment Summary:	For lipid extraction, cell pellets were mixed with 200 µL of cold (-20°C) methanol:water (1:1, v/v) and sonicated with an ultrasonic homogenizer (UP200S, Hielscher Ultrasound Technology, HIELSCHER GmbH, Chamerau, Germany) for 16 bursts (0.5 second pulse) at 80% amplitude. Homogenates (100 µL) were mixed with 320 µL of cold (-20°C) methanol containing 1.6 ppm of sphinganine (d17:0) as the internal standard. Samples were then vortex-mixed for 2 minutes, followed by the addition of 80 µL of methyl tert-butyl ether. Subsequently, samples were vortex-mixed (1 hour, RT). After centrifugation (16,000 × g, 15°C, 10 minutes), samples were used for ultra-high performance liquid chromatography (UHPLC; Agilent 1290 Infinity II, Agilent Technologies Inc., Santa Clara, CA) coupled with (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) (Agilent 6546): 100 µL of each sample was divided between two UHPLC-MS vials with inserts (50 µL/each) for direct injection into the system for LC-MS analyses in positive and negative ionization modes.

Treatment ID:

TR002248

Treatment Summary:

For lipid extraction, cell pellets were mixed with 200 µL of cold (-20°C) methanol:water (1:1, v/v) and sonicated with an ultrasonic homogenizer (UP200S, Hielscher Ultrasound Technology, HIELSCHER GmbH, Chamerau, Germany) for 16 bursts (0.5 second pulse) at 80% amplitude. Homogenates (100 µL) were mixed with 320 µL of cold (-20°C) methanol containing 1.6 ppm of sphinganine (d17:0) as the internal standard. Samples were then vortex-mixed for 2 minutes, followed by the addition of 80 µL of methyl tert-butyl ether. Subsequently, samples were vortex-mixed (1 hour, RT). After centrifugation (16,000 × g, 15°C, 10 minutes), samples were used for ultra-high performance liquid chromatography (UHPLC; Agilent 1290 Infinity II, Agilent Technologies Inc., Santa Clara, CA) coupled with (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) (Agilent 6546): 100 µL of each sample was divided between two UHPLC-MS vials with inserts (50 µL/each) for direct injection into the system for LC-MS analyses in positive and negative ionization modes.

Sample Preparation:

Sampleprep ID:	SP002242
Sampleprep Summary:	For lipid extraction from Jurkat and T cell blasts, cell pellets were mixed with 200 µL of cold (-20°C) methanol:water (1:1, v/v) and sonicated with an ultrasonic homogenizer (UP200S, Hielscher Ultrasound Technology, HIELSCHER GmbH, Chamerau, Germany) for 16 bursts (0.5 second pulse) at 80% amplitude. Homogenates (100 µL) were mixed with 320 µL of cold (-20°C) methanol containing 1.6 ppm of sphinganine (d17:0) as the internal standard. Samples were then vortex-mixed for 2 minutes, followed by the addition of 80 µL of methyl tert-butyl ether. Subsequently, samples were vortex-mixed (1 hour, RT). After centrifugation (16,000 × g, 15°C, 10 minutes), samples were used for ultra-high performance liquid chromatography (UHPLC; Agilent 1290 Infinity II, Agilent Technologies Inc., Santa Clara, CA) coupled with (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) (Agilent 6546): 100 µL of each sample was divided between two UHPLC-MS vials with inserts (50 µL/each) for direct injection into the system for LC-MS analyses in positive and negative ionization modes.

Sampleprep ID:

SP002242

Sampleprep Summary:

For lipid extraction from Jurkat and T cell blasts, cell pellets were mixed with 200 µL of cold (-20°C) methanol:water (1:1, v/v) and sonicated with an ultrasonic homogenizer (UP200S, Hielscher Ultrasound Technology, HIELSCHER GmbH, Chamerau, Germany) for 16 bursts (0.5 second pulse) at 80% amplitude. Homogenates (100 µL) were mixed with 320 µL of cold (-20°C) methanol containing 1.6 ppm of sphinganine (d17:0) as the internal standard. Samples were then vortex-mixed for 2 minutes, followed by the addition of 80 µL of methyl tert-butyl ether. Subsequently, samples were vortex-mixed (1 hour, RT). After centrifugation (16,000 × g, 15°C, 10 minutes), samples were used for ultra-high performance liquid chromatography (UHPLC; Agilent 1290 Infinity II, Agilent Technologies Inc., Santa Clara, CA) coupled with (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) (Agilent 6546): 100 µL of each sample was divided between two UHPLC-MS vials with inserts (50 µL/each) for direct injection into the system for LC-MS analyses in positive and negative ionization modes.

Combined analysis:

Analysis ID	AN003520	AN003521
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	Agilent InfinityLab Poroshell 120 EC–C18, 3.0 × 5 mm, 2.7 μm	Agilent InfinityLab Poroshell 120 EC–C18, 3.0 × 5 mm, 2.7 μm
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6546 QTOF	Agilent 6546 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	AREA	AREA

Chromatography:

Chromatography ID:	CH002599
Chromatography Summary:	RP-UHPLC-ESI(+)-QTOF MS
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent InfinityLab Poroshell 120 EC–C18, 3.0 × 5 mm, 2.7 μm
Chromatography Type:	Reversed phase

Chromatography ID:	CH002600
Chromatography Summary:	RP-UHPLC-ESI(-)-QTOF MS
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent InfinityLab Poroshell 120 EC–C18, 3.0 × 5 mm, 2.7 μm
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003278
Analysis ID:	AN003520
Instrument Name:	Agilent 6546 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The Agilent 6545 QTOF mass spectrometer equipped with a dual AJS ESI ion source was set with the following parameters: 150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 50 to 1800 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds were used throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 for the positive and m/z 119.0363 for the negative ionization modes; and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098 for the positive and m/z 980.0163 (HP-0921+acetate) for the negative ionization modes. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. Ten Iterative-MS/MS runs were performed for both ion modes at the end of the analytical run. They were operated with an MS and MS/MS scan rates of 3 spectra/s, 40–1800 m/z mass window, a narrow (∼ 1.3 amu) MS/MS isolation width, 3 precursors per cycle, and 5000 counts and 0.001% of MS/MS threshold. Five iterative-MS/MS runs were set with a collision energy of 20 eV, and the subsequent five runs were performed at 40 eV. References masses and contaminants detected in blank samples were excluded from the analysis to avoid inclusion in the iterative-MS/MS.
Ion Mode:	POSITIVE

MS ID:	MS003279
Analysis ID:	AN003521
Instrument Name:	Agilent 6546 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The Agilent 6545 QTOF mass spectrometer equipped with a dual AJS ESI ion source was set with the following parameters: 150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 50 to 1800 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds were used throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 for the positive and m/z 119.0363 for the negative ionization modes; and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098 for the positive and m/z 980.0163 (HP-0921+acetate) for the negative ionization modes. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. Ten Iterative-MS/MS runs were performed for both ion modes at the end of the analytical run. They were operated with an MS and MS/MS scan rates of 3 spectra/s, 40–1800 m/z mass window, a narrow (∼ 1.3 amu) MS/MS isolation width, 3 precursors per cycle, and 5000 counts and 0.001% of MS/MS threshold. Five iterative-MS/MS runs were set with a collision energy of 20 eV, and the subsequent five runs were performed at 40 eV. References masses and contaminants detected in blank samples were excluded from the analysis to avoid inclusion in the iterative-MS/MS.
Ion Mode:	NEGATIVE