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MB Sample ID: SA214100
Local Sample ID: | 2_GAT_3-pos |
Subject ID: | SU002326 |
Subject Type: | Plant |
Subject Species: | Arabidopsis thaliana |
Taxonomy ID: | 3702 |
Age Or Age Range: | 10 day old seedlings |
Species Group: | Roots |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002326 |
Subject Type: | Plant |
Subject Species: | Arabidopsis thaliana |
Taxonomy ID: | 3702 |
Age Or Age Range: | 10 day old seedlings |
Species Group: | Roots |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
2_GAT_3-pos | SA214100 | FL026011 | gat1_2.1 (mutant) | Tissue type |
2_GAT_3-pos | SA214100 | FL026011 | 2 | Time (hours) |
Collection:
Collection ID: | CO002319 |
Collection Summary: | For the metabolomics study using dual-isotope labeling, wildtype Arabidopsis ecotype Columbia seeds were grown on vertical plates at 22°C under continuous light (ca. 70 µmol m-2 s-1), on a defined nutrient medium previously described11. The medium consisted of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar. Ten-day old seedlings were transferred to plates containing the same medium, except the nitrogen source was replaced with 10 mM [13C5,15N2]glutamine. Root tissue was excised after exposure to medium containing labeled glutamine for 2, 4, 6 and 8h to represent time course incorporation of carbon and nitrogen into metabolism. Untreated roots were used as unlabeled (0h) controls. Each plate yielded ~100 mg of root tissue and served as a single replicate. Four replicates per sample type were collected and flash frozen using liquid N2 for total metabolite extraction. |
Sample Type: | Plant |
Collection Method: | Flash frozen in Liquid N2 |
Collection Location: | Donald Danforth Plant Science Center |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR002338 |
Treatment Summary: | For the metabolomics study using dual-isotope labeling, wildtype Arabidopsis ecotype Columbia seeds were grown on vertical plates at 22°C under continuous light (ca. 70 µmol m-2 s-1), on a defined nutrient medium previously described11. The medium consisted of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar. Ten-day old seedlings were transferred to plates containing the same medium, except the nitrogen source was replaced with 10 mM [13C5,15N2]glutamine. Root tissue was excised after exposure to medium containing labeled glutamine for 2, 4, 6 and 8h to represent time course incorporation of carbon and nitrogen into metabolism. Untreated roots were used as unlabeled (0h) controls. Each plate yielded ~100 mg of root tissue and served as a single replicate. Four replicates per sample type were collected and flash frozen using liquid N2 for total metabolite extraction. |
Sample Preparation:
Sampleprep ID: | SP002332 |
Sampleprep Summary: | Frozen Arabidopsis root tissue was homogenized using a tissue lyser, and extraction was carried out using 1 mL of 4:1 methanol: water (v/v) with incubation in an ultra-sonication bath for 30 min followed by shaking for 30 min at 4°C. The mixture was then centrifuged at 21,000 x g for 10 min at 4°C; supernatant was transferred into fresh tubes and evaporated to dryness using a speedvac centrifuge at ambient temperature. Dried residue was re-suspended in 200 µL of 1:1 methanol: water (v/v), filtered using 0.2 µm PTFE micro centrifuge filters and transferred to glass vials for HILIC-HRMS runs. |
Processing Storage Conditions: | -80℃ |
Extraction Method: | 4:1 Methanol:Water |
Combined analysis:
Analysis ID | AN003657 | AN003658 |
---|---|---|
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: | CH002710 |
Chromatography Summary: | Chromatographic separation using HILIC was achieved using an Agilent 1290 Infinity II UHPLC system equipped with a SeQuant® ZIC®-HILIC (100 x 2.1 x 3.5 µm) column (EMD Millipore, Burlington, MA). Mobile phases A and B were comprised of 5 mM ammonium acetate (pH 4.0) in water and 90% acetonitrile with 0.1 % acetic acid, respectively. A flow rate of 0.3 mL min-1 was used to elute compounds with the following gradient: 87% B for 5 minutes, decreased to 55% B over the next 8 minutes and held for 2.5 minutes before returning to 87% and equilibrating the column for 3 minutes. |
Instrument Name: | Agilent 1290 Infinity II |
Column Name: | SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) |
Column Temperature: | 40 |
Flow Rate: | 0.3 mL min-1 |
Internal Standard: | Equisplash |
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: | MS003408 |
Analysis ID: | AN003657 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The heated electrospray ionization (HESI) conditions used were as follows; 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%. Full MS data were collected using a Q-Exactive Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific) in both positive and negative ionization mode separately from mass ranges 75-1100 m/z and 65-900 m/z, respectively, at 140,000 resolution. The automatic gain control (AGC) was set to 3 x 106 and maximum injection time (IT) used was 524 ms. |
Ion Mode: | POSITIVE |
MS ID: | MS003409 |
Analysis ID: | AN003658 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The heated electrospray ionization (HESI) conditions used were as follows; 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%. Full MS data were collected using a Q-Exactive Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific) in both positive and negative ionization mode separately from mass ranges 75-1100 m/z and 65-900 m/z, respectively, at 140,000 resolution. The automatic gain control (AGC) was set to 3 x 106 and maximum injection time (IT) used was 524 ms. |
Ion Mode: | NEGATIVE |