Return to study ST001812 main page
MB Sample ID: SA168274
Local Sample ID: | E_15NGln_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 |
Select appropriate tab below to view additional metadata details:
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_3_neg | SA168274 | FL020038 | E_15NGln_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]. |
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. |
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 |