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MB Sample ID: SA071870
Local Sample ID: | B5_45 |
Subject ID: | SU001106 |
Subject Type: | Plant |
Subject Species: | Arabidopsis thaliana |
Taxonomy ID: | 3702 |
Age Or Age Range: | 3-4 weeks |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|
B5_45 | SA071870 | FL011266 | SALK_108849C_KO | Genotype |
B5_45 | SA071870 | FL011266 | 1/2MS-media_3weeks | Treatment |
B5_45 | SA071870 | 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. |
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 |