Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA214119

Local Sample ID:	6_GAT_1-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
6_GAT_1-pos	SA214119	FL026013	gat1_2.1 (mutant)	Tissue type
6_GAT_1-pos	SA214119	FL026013	6	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.

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