Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA168282

Local Sample ID:	E_15NGln_D_3_neg
Subject ID:	SU001889
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:	SU001889
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
E_15NGln_D_3_neg	SA168282	FL020046	E_15NGln_D_3_neg	Rawfilename

Collection:

Collection ID:	CO001882
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:	TR001902
Treatment Summary:	Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for growth. 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].

Treatment ID:

TR001902

Treatment Summary:

Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for growth. 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].

Sample Preparation:

Sampleprep ID:	SP001895
Sampleprep Summary:	Fifty mg of root tissue was excised from 10 day old seedlings of Arabidopsis grown with 5 mM KNO3, 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. Dried metabolite extracts were re-suspended in HEPES buffer pH 7.5 instead of 1:1 methanol:water. Samples were spiked with a final concentration of 1 µM 15N Gln and 5 µg of the full length or glutaminase domain versions of recombinant GAT1_2.1 protein along with 2 mM DTT and 5 mM MgCl2. Following this, samples were incubated at 37 °C for 2 hours and then filtered through a 3K micro centrifuge filter (Sigma-Aldrich) to remove the protein. Samples were then evaporated to dryness using a vacufuge at ambient temperature and the residue was re-dissolved in 1:1 methanol:water, filtered with a 0.2 µm PTFE microfuge filters (Whatman) and subjected to LC-MS analysis and ammonium quantification.

Sampleprep ID:

SP001895

Sampleprep Summary:

Fifty mg of root tissue was excised from 10 day old seedlings of Arabidopsis grown with 5 mM KNO3, 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. Dried metabolite extracts were re-suspended in HEPES buffer pH 7.5 instead of 1:1 methanol:water. Samples were spiked with a final concentration of 1 µM 15N Gln and 5 µg of the full length or glutaminase domain versions of recombinant GAT1_2.1 protein along with 2 mM DTT and 5 mM MgCl2. Following this, samples were incubated at 37 °C for 2 hours and then filtered through a 3K micro centrifuge filter (Sigma-Aldrich) to remove the protein. Samples were then evaporated to dryness using a vacufuge at ambient temperature and the residue was re-dissolved in 1:1 methanol:water, filtered with a 0.2 µm PTFE microfuge filters (Whatman) and subjected to LC-MS analysis and ammonium quantification.

Combined analysis:

Analysis ID	AN002937	AN002938
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	Intensity	Intensity

Chromatography:

Chromatography ID:	CH002176
Methods Filename:	Enzyme_spike_analysis.pdf
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:	MS002728
Analysis ID:	AN002937
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Full MS measurements were collected from mass ranges of 75-1100 m/z and 65-900 m/z in positive and negative ionization modes respectively at 140,000 resolutions. The AGC target and maximum IT was set to 3 e6 and 524 ms respectively.
Ion Mode:	POSITIVE
Capillary Temperature:	250
Capillary Voltage:	3.9
Analysis Protocol File:	Enzyme_spike_analysis.pdf

MS ID:	MS002729
Analysis ID:	AN002938
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Full MS measurements were collected from mass ranges of 75-1100 m/z and 65-900 m/z in positive and negative ionization modes respectively at 140,000 resolutions. The AGC target and maximum IT was set to 3 e6 and 524 ms respectively.
Ion Mode:	NEGATIVE
Capillary Temperature:	250
Capillary Voltage:	3.5
Analysis Protocol File:	Enzyme_spike_analysis.pdf