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.
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 | ST002365 |
| Study Title | [U-13C]glutamine tracing in activated WT, GOT1 or GLUD1 knockout CD8+ T cells |
| Study 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. |
| wxu29@jhmi.edu | |
| Phone | 443-220-9936 |
| Submit Date | 2022-11-28 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | d |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-12-15 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA237049 | Glud1_KO_01 | GLUD1_KO |
| SA237050 | Glud1_KO_02 | GLUD1_KO |
| SA237051 | Glud1_KO_03 | GLUD1_KO |
| SA237052 | Glud1_Wt_03 | GLUD1_WT |
| SA237053 | Glud1_Wt_02 | GLUD1_WT |
| SA237054 | Glud1_Wt_01 | GLUD1_WT |
| SA237055 | Got1_KO_01 | GOT1_KO |
| SA237056 | Got1_KO_02 | GOT1_KO |
| SA237057 | Got1_KO_03 | GOT1_KO |
| SA237058 | Got1_Wt_02 | GOT1_WT |
| SA237059 | Got1_Wt_03 | GOT1_WT |
| SA237060 | Got1_Wt_01 | GOT1_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 |
