Summary of Study ST002365

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 PR001520. The data can be accessed directly via it's Project DOI: 10.21228/M8713J This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST002365
Study Title[U-13C]glutamine tracing in activated WT, GOT1 or GLUD1 knockout CD8+ T cells
Study SummaryWT, GOT1 knockout or GLUD1 knockout CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were pulsed with [U-13C]glutamine for 4-6 hours. Intracellular glutamine-derived glutamate and α-ketoglutarate levels were quantified using MS.
Institute
Johns Hopkins University
Last NameXu
First NameWei
Address1650 Orleans Street, Baltimore, MD 21287, USA.
Emailwxu29@jhmi.edu
Phone443-220-9936
Submit Date2022-11-28
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2022-12-15
Release Version1
Wei Xu Wei Xu
https://dx.doi.org/10.21228/M8713J
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001520
Project DOI:doi: 10.21228/M8713J
Project Title:[U-13C]glutamine tracing in activated WT or GOT1 or GLUD1 knockout CD8+ T cells
Project Type:MS quantifying intracellular glutamate levels
Project Summary:WT, GOT1 knockout or GLUD1 knockout CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were pulsed with [U-13C]glutamine for 4-6 hours. Intracellular glutamine-derived glutamate and α-ketoglutarate levels were quantified using MS.
Institute:Johns Hopkins University
Last Name:Xu
First Name:Wei
Address:1650 Orleans Street, Baltimore, MD 21287, USA.
Email:wxu29@jhmi.edu
Phone:443-220-9936

Subject:

Subject ID:SU002454
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:6-8 weeks
Gender:Male and female

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA237049Glud1_KO_01GLUD1_KO
SA237050Glud1_KO_02GLUD1_KO
SA237051Glud1_KO_03GLUD1_KO
SA237052Glud1_Wt_03GLUD1_WT
SA237053Glud1_Wt_02GLUD1_WT
SA237054Glud1_Wt_01GLUD1_WT
SA237055Got1_KO_01GOT1_KO
SA237056Got1_KO_02GOT1_KO
SA237057Got1_KO_03GOT1_KO
SA237058Got1_Wt_02GOT1_WT
SA237059Got1_Wt_03GOT1_WT
SA237060Got1_Wt_01GOT1_WT
Showing results 1 to 12 of 12

Collection:

Collection ID:CO002447
Collection Summary:Cells were spun down and washed once with pre-warmed PBS and metabolites were immediately extracted or stored at -80℃ until further extraction.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR002466
Treatment Summary:CD8+ T cells were isolated from spleens and lymph nodes from WT, T cell conditional GOT1 or GLUD1 knockout mice. Cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were counted and resuspended in full media containing 4 mM [U-13C]glutamine at 2 E6 mL-1. Normal FBS was substituted with dialyzed FBS. Cells were collected for LC-MS analysis 4-6 hrs post incubation.

Sample Preparation:

Sampleprep ID:SP002460
Sampleprep Summary:Cells were spun down and washed once with pre-warmed PBS and metabolites were immediately extracted by adding methanol:water (80:20, v/v) extraction solution, sonicated and stored at -80 °C for at least 2 hrs to precipitate the proteins. Supernatant after centrifugation at 14,000xg for 10 minutes was dried under nitrogen gas. Metabolites were then reconstituted using ACN:water (50:50, v/v) overnight at 4 °C. Soluble metabolites after centrifugation at 14,000xg for 10 minutes were subjected to analysis by liquid chromatography mass spectrometry (LC-MS).

Combined analysis:

Analysis ID AN003860
Analysis type MS
Chromatography type Ion pair
Chromatography system Agilent 1290 Infinity
Column Agilent Zorbax Extend C18 (150 x 2.1mm,1.8 um)
MS Type ESI
MS instrument type QTOF
MS instrument name Agilent 6520 QTOF
Ion Mode NEGATIVE
Units AUC

Chromatography:

Chromatography ID:CH002858
Instrument Name:Agilent 1290 Infinity
Column Name:Agilent Zorbax Extend C18 (150 x 2.1mm,1.8 um)
Chromatography Type:Ion pair

MS:

MS ID:MS003601
Analysis ID:AN003860
Instrument Name:Agilent 6520 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:The optimized ESI Q-TOF parameters for MS experiments were: ion polarity, negative; gas temperature, 325 °C; drying gas, 10 L min-1; nebulizer pressure, 45 psig; capillary voltage, 4,000 V; fragmentor, 140 V; skimmer, 65 V; mass range, 50-1100 m/z; acquisition rate, 1.5 spectra s-1; instrument state, extended dynamic range (1700 m/z, 2 GHz). Spectra were internally mass calibrated in real time by continuous infusion of a reference mass solution using an isocratic pump connected to a dual sprayer feeding into an electrospray ionization source. Data were acquired with MassHunter Acquisition software. A metabolite database with retention times based on the ion-pairing method was developed using Agilent MassHunter PCDL manager software. The isotopologue peak extractions were achieved by Agilent MassHunter Profinder software.
Ion Mode:NEGATIVE
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