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MB Sample ID: SA168238
Local Sample ID: | GATp2GLU-3-neg-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 |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|
GATp2GLU-3-neg-PRM | SA168238 | FL020002 | GATp2GLU-3-neg-PRM | Raw file name |
GATp2GLU-3-neg-PRM | SA168238 | FL020002 | Positive Control | SampleType |
GATp2GLU-3-neg-PRM | SA168238 | FL020002 | gat1_2.1 | 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. |
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. |
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 |