Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA237056

Local Sample ID:	Got1_KO_02
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

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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:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Got1_KO_02	SA237056	FL029357	GOT1_KO	Treatment

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.

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).

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