Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA071868

Local Sample ID:	B3_43
Subject ID:	SU001106
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702
Age Or Age Range:	3-4 weeks

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Subject:

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

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
B3_43	SA071868	FL011266	SALK_108849C_KO	Genotype
B3_43	SA071868	FL011266	1/2MS-media_3weeks	Treatment
B3_43	SA071868	FL011266	Whole Plant	Tissue Type

Collection:

Collection ID:	CO001100
Collection Summary:	For metabolic analysis, samples were immediately frozen in liquid nitrogen, powdered, lyophilized, and stored at -80°C until analysis.
Sample Type:	Plant

Treatment:

Treatment ID:	TR001120
Treatment Summary:	Plants of either wild type or knockout genotype were grown on 1/2 MS media for 3-4 weeks, or grown on soil for 4 weeks.
Plant Light Period:	12:12h

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.

Combined analysis:

Analysis ID	AN001736	AN001737	AN001738
Analysis type	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE
Units	AU	AU	AU

Chromatography:

Chromatography ID:	CH001228
Chromatography Summary:	HILIC chromatography 17 minute run. Positive and Negative mode.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	The gradient started at 100% B for 2 minutes, then brought to 70% B by 7.7 minutes, 40% B by 9.5 minutes, back to 100% B by 12.75 minutes and held at 100% B until 17 minutes.
Flow Rate:	0.4 mL/min
Internal Standard:	5ug Val-Tyr-Val+
Sample Injection:	5uL
Solvent A:	100% water; 0.125% formic acid; 10 mM ammonium formate
Solvent B:	95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
Washing Buffer:	Water:ACN 50:50
Target Sample Temperature:	4
Chromatography Type:	HILIC

Chromatography ID:	CH001229
Chromatography Summary:	CSH C18 lipidomics run. Positive mode. As in Cajka et al. 2017 "Validating Quantitative untargeted lipidomics across nine liquid chromatography-High-Resolution Mass spectrometry platforms"
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	65
Flow Gradient:	: 0 min 15% (B); 0−2 min 30% (B); 2−2.5 min 48% (B); 2.5−11 min 82% (B); 11−11.5 min 99% (B); 11.5−12 min 99% (B); 12−12.1 min 15% (B); and 12.1−15 min 15% (B)
Flow Rate:	0.6 mL/min
Internal Standard:	[LPE(17:1), LPC(17:0), PC(12:0/13:0), PE(17:0/17:0), PG(17:0/17:0), d7-cholesterol, SM(d18:1/17:0), Cer(d18:1/17:0), sphingosine (d17:1), DG(12:0/12:0/0:0), DG(18:1/2:0/0:0), and d5-TG- (17:0/17:1/17:0)]
Sample Injection:	3uL
Solvent A:	60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Washing Buffer:	IPA
Target Sample Temperature:	4
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001605
Analysis ID:	AN001736
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
Ion Mode:	POSITIVE
Collision Energy:	NCE 20.30.40
Ionization:	Positive
Scan Range Moverz:	60-900
Scanning Cycle:	Top 4 ions selected for MS/MS

MS ID:	MS001606
Analysis ID:	AN001737
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
Ion Mode:	NEGATIVE
Collision Energy:	NCE 20.30.40
Ionization:	Negative
Scan Range Moverz:	60-900
Scanning Cycle:	Top 4 ions selected for MS/MS

MS ID:	MS001607
Analysis ID:	AN001738
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
Ion Mode:	POSITIVE
Collision Energy:	NCE 20.30.40
Ionization:	Positive
Scan Range Moverz:	120-1200
Scanning Cycle:	Top 4 ions selected for MS/MS