Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA205958

Local Sample ID:	Jurkat_SMasa_3
Subject ID:	SU002236
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002236
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Jurkat_SMasa_3	SA205958	FL025274	CASE	Factor1

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