Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA168267

Local Sample ID:	WTp2GLU-2-pos-PRM
Subject ID:	SU001888
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702
Genotype Strain:	Col-0
Age Or Age Range:	10 day old seedlings
Gender:	Not applicable

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Subject:

Subject ID:	SU001888
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702
Genotype Strain:	Col-0
Age Or Age Range:	10 day old seedlings
Gender:	Not applicable

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
WTp2GLU-2-pos-PRM	SA168267	FL020031	WTp2GLU-2-pos-PRM	Raw file name
WTp2GLU-2-pos-PRM	SA168267	FL020031	Positive Control	SampleType
WTp2GLU-2-pos-PRM	SA168267	FL020031	Wildtype	Genotype

Collection:

Collection ID:	CO001881
Collection Summary:	Roots from 50 seedlings grown in plates containing required treatment were collected and processed as single replicate.
Collection Protocol ID:	001
Sample Type:	Plant
Collection Method:	50 mg collected and flash frozen in Liquid N2
Collection Location:	London Research and Development Center
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001901
Treatment Summary:	Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for Gln and Glu treatments. Plants were grown on vertical plates at 22 °C under continuous light (ca. 70 μmol m-2 s-2), as previously described by Ivanov et al. (2012) on a defined nutrient medium containing a final concentration of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 125 μg FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar [28]. Ten-day old seedlings were transferred to plates containing the same medium without nitrogen as control or 10 mM Gln as sole N source. After 2 h, root tissue was harvested, frozen in liquid N2 and stored at -80 °C until total metabolite extractions was carried out. For growth in Gln and Glu, the same media and growth conditions were used with the exception of 5 mM KNO3 being substituted with either 2 mM Gln or 2 mM Glu and tissue was collected after 10 days.

Treatment ID:

TR001901

Treatment Summary:

Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for Gln and Glu treatments. Plants were grown on vertical plates at 22 °C under continuous light (ca. 70 μmol m-2 s-2), as previously described by Ivanov et al. (2012) on a defined nutrient medium containing a final concentration of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 125 μg FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar [28]. Ten-day old seedlings were transferred to plates containing the same medium without nitrogen as control or 10 mM Gln as sole N source. After 2 h, root tissue was harvested, frozen in liquid N2 and stored at -80 °C until total metabolite extractions was carried out. For growth in Gln and Glu, the same media and growth conditions were used with the exception of 5 mM KNO3 being substituted with either 2 mM Gln or 2 mM Glu and tissue was collected after 10 days.

Sample Preparation:

Sampleprep ID:	SP001894
Sampleprep Summary:	Fifty mg of root tissue was excised from 10 day old seedlings of WT or gat1_2.1 grown under conditions described above, collected in 2 mL Eppendorf tubes and flash frozen in liquid N2. Frozen tissue was homogenized using a tissue lyser and metabolites were isolated using 1 mL of methanol:water (4:1) with incubation in an ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C. The mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of the supernatant was transferred into fresh tubes and evaporated to dryness using a Vacufuge at ambient temperature. The residue was re-dissolved in 500 µl of 1:1 methanol:water and the samples were filtered using a 0.2 µm PTFE microfuge filter (Cytiva Whatman). Five µl of 1 µg/mL 13C6 Phe was added to the samples for monitoring the quality of LC-MS runs.

Sampleprep ID:

SP001894

Sampleprep Summary:

Fifty mg of root tissue was excised from 10 day old seedlings of WT or gat1_2.1 grown under conditions described above, collected in 2 mL Eppendorf tubes and flash frozen in liquid N2. Frozen tissue was homogenized using a tissue lyser and metabolites were isolated using 1 mL of methanol:water (4:1) with incubation in an ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C. The mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of the supernatant was transferred into fresh tubes and evaporated to dryness using a Vacufuge at ambient temperature. The residue was re-dissolved in 500 µl of 1:1 methanol:water and the samples were filtered using a 0.2 µm PTFE microfuge filter (Cytiva Whatman). Five µl of 1 µg/mL 13C6 Phe was added to the samples for monitoring the quality of LC-MS runs.

Combined analysis:

Analysis ID	AN002935	AN002936
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	umol g FW-1	umol g FW-1

Chromatography:

Chromatography ID:	CH002175
Methods Filename:	Targeted Metabolite Analysis
Instrument Name:	Agilent 1290 Infinity II
Column Name:	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:	35
Flow Rate:	0.3 mL min-1
Internal Standard:	13C6 Phenylalanine
Solvent A:	100% water; 5 mM ammonium acetate, pH 4
Solvent B:	90% acetonitrile/10% water; 0.1% acetic acid
Chromatography Type:	HILIC

MS:

MS ID:	MS002726
Analysis ID:	AN002935
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The following heated electrospray ionization (HESI) conditions were optimized for the analysis of amino and organic acids: spray voltage, 3.9 kV (ESI+), 3.5 kV (ESI-); capillary temperature, 250 °C; probe heater temperature, 450 °C; sheath gas, 30 arbitrary units; auxiliary gas, 8 arbitrary units; and S-Lens RF level, 60%. Injections of 5 μl were used with a flow rate of 0.3 mL min-1. Compounds were detected and monitored using targeted MS/MS, spectra were collected at 17,500 resolution, AGC target 1e6, maximum IT 65 ms, isolation window of 1 m/z, normalized collision energy of 30, intensity threshold of 1.6e5 and 10s dynamic exclusion.
Ion Mode:	POSITIVE
Analysis Protocol File:	Targeted_Metabolite_Analysis.pdf

MS ID:	MS002727
Analysis ID:	AN002936
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The following heated electrospray ionization (HESI) conditions were optimized for the analysis of amino and organic acids: spray voltage, 3.9 kV (ESI+), 3.5 kV (ESI-); capillary temperature, 250 °C; probe heater temperature, 450 °C; sheath gas, 30 arbitrary units; auxiliary gas, 8 arbitrary units; and S-Lens RF level, 60%. Injections of 5 μl were used with a flow rate of 0.3 mL min-1. Compounds were detected and monitored using targeted MS/MS, spectra were collected at 17,500 resolution, AGC target 1e6, maximum IT 65 ms, isolation window of 1 m/z, normalized collision energy of 30, intensity threshold of 1.6e5 and 10s dynamic exclusion.
Ion Mode:	NEGATIVE
Analysis Protocol File:	Targeted_Metabolite_Analysis