Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA071874

Local Sample ID:	D2_20
Subject ID:	SU001106
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702
Age Or Age Range:	3-4 weeks

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Sample Preparation:

Sampleprep ID:	SP001113
Sampleprep Summary:	Samples (10 mg lyophilized plant tissue) were extracted using biphasic extraction technique adapted from Matyash et al. 2008. In summary 225 µL LC-MS grade methanol was added to lyophilized plant tissue in 2 mL Eppendorf tube, vortexed for 10 seconds, followed by addition of 750 µL methyl tert-butyl ether (MTBE). Each sample was then vortexed for 10 seconds, shook on orbital shaker at maximum speed for six minutes, followed by addition of 188 µL LC-MS grade water. Finally, each sample was vortexed for 10 seconds and centrifuged for 2 minutes at 14,000 rpm. The resultant two-phase extract was aliquoted into four clean 1.5 mL Eppendorf tubes. The result was two 350 µL aliquots of MTBE phase (top), and two 110 µL aliquots of methanol/water phase (bottom). Extraction was carried out at 4°C. All extract tubes were dried under vacuum and frozen at -80°C until LC-MS/MS analysis. Lipidomics analysis followed methods of Cajka et al. 2017. Briefly, extract from one aliquot of MTBE phase was resuspended in 110 µL methanol/toluene (9:1, v/v), vortexed for 10 seconds, centrifuged for 2 minutes at 14,000 rpm, transferred to HPLC vial, and stored at 4°C until LC-MS/MS analysis. Gradient, internal standards, mobile phases, and data collection methods were identical to Cajka et al. 2017. Sample injection volume was 3 µL on a Waters Acquity UPLC CSH C18 column (100mm x 2.1mm, 1.7 μm particle size). A Thermo Vanquish Focused UHPLC system coupled to Thermo Q Exactive HF mass spectrometer was used for all untargeted analysis. Untargeted polar metabolomics analysis followed methods of Niehaus et al. 2018. Briefly, one aliquot of methanol/water phase extract was resuspended in 100 µL acetonitrile/water (4:1 v/v) with internal standards including 5 µg/ml Val-Tyr-Val. Analysis was carried out using Waters Acquity UPLC BEH Amide (150mm x 2.1 mm id, 1.7 μm particle size) column with identical mobile phase, gradient, injection volume, and data collection to Niehaus et al. 2018.

Sampleprep ID:

SP001113

Sampleprep Summary:

Samples (10 mg lyophilized plant tissue) were extracted using biphasic extraction technique adapted from Matyash et al. 2008. In summary 225 µL LC-MS grade methanol was added to lyophilized plant tissue in 2 mL Eppendorf tube, vortexed for 10 seconds, followed by addition of 750 µL methyl tert-butyl ether (MTBE). Each sample was then vortexed for 10 seconds, shook on orbital shaker at maximum speed for six minutes, followed by addition of 188 µL LC-MS grade water. Finally, each sample was vortexed for 10 seconds and centrifuged for 2 minutes at 14,000 rpm. The resultant two-phase extract was aliquoted into four clean 1.5 mL Eppendorf tubes. The result was two 350 µL aliquots of MTBE phase (top), and two 110 µL aliquots of methanol/water phase (bottom). Extraction was carried out at 4°C. All extract tubes were dried under vacuum and frozen at -80°C until LC-MS/MS analysis. Lipidomics analysis followed methods of Cajka et al. 2017. Briefly, extract from one aliquot of MTBE phase was resuspended in 110 µL methanol/toluene (9:1, v/v), vortexed for 10 seconds, centrifuged for 2 minutes at 14,000 rpm, transferred to HPLC vial, and stored at 4°C until LC-MS/MS analysis. Gradient, internal standards, mobile phases, and data collection methods were identical to Cajka et al. 2017. Sample injection volume was 3 µL on a Waters Acquity UPLC CSH C18 column (100mm x 2.1mm, 1.7 μm particle size). A Thermo Vanquish Focused UHPLC system coupled to Thermo Q Exactive HF mass spectrometer was used for all untargeted analysis. Untargeted polar metabolomics analysis followed methods of Niehaus et al. 2018. Briefly, one aliquot of methanol/water phase extract was resuspended in 100 µL acetonitrile/water (4:1 v/v) with internal standards including 5 µg/ml Val-Tyr-Val. Analysis was carried out using Waters Acquity UPLC BEH Amide (150mm x 2.1 mm id, 1.7 μm particle size) column with identical mobile phase, gradient, injection volume, and data collection to Niehaus et al. 2018.