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MB Sample ID: SA099828
| Local Sample ID: | MAD3_NEG C |
| Subject ID: | SU001445 |
| Subject Type: | Plant |
| Subject Species: | Quercus ilex |
| Taxonomy ID: | 58334 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001445 |
| Subject Type: | Plant |
| Subject Species: | Quercus ilex |
| Taxonomy ID: | 58334 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| MAD3_NEG C | SA099828 | FL014123 | MAD | Mixture |
Collection:
| Collection ID: | CO001440 |
| Collection Summary: | Sample collection: Mature acorns from holm oak (Quercus ilex L. subsp.ballota [Desf.] Samp.) were collected from four different trees located in Aldea de Cuenca (province of Cordoba, Andalusia, Spain). All acorns were picked at the optimal harvest maturity window on the same day. Sorting, disinfection, and storage of healthy acorns were conducted according to Bonner & Vozzo, (1987). Acorn flour preparation: Healthy acorns (20 units per tree) were scarified with a knife by making transversal and longitudinal cuts, thus permitting the pericarp to be rapidly removed. Flour was prepared by seed (without seed coat) grinding with liquid nitrogen in a blade mill (IKA Dry Mill Basic A10) until a powder was obtained (Valero Galván, Jorrín Novo, Cabrera, et al., 2012). Flour was lyophilized and then macerated in a mortar until a fine powder was obtained. Samples were stored at 4 ºC in within a desiccator, in darkness, until NIRS analysis or metabolite extraction. |
| Sample Type: | Acorns |
| Storage Conditions: | Room temperature |
Treatment:
| Treatment ID: | TR001460 |
| Treatment Summary: | Mature acorns from holm oak (Quercus ilex L. subsp.ballota [Desf.] Samp.) were collected from nine different trees. From these, four were selected for metabolomic analysis (POL, BOL, NTC, and MAD). |
| Plant Growth Location: | Aldea de Cuenca (province of Cordoba, Andalusia, Spain) |
Sample Preparation:
| Sampleprep ID: | SP001453 |
| Sampleprep Summary: | Metabolites were extracted from acorn flour as described by Valledor et al., (2014), with minor modifications. 600 µL of ice-cold methanol: chloroform: water (5:2:2) was added to 50 mg of acorn flour, mixed by vortexing, and the mixture sonicated (ultrasonic bath, 40 kHZ for 10 min). After centrifugation (4 oC, 4 min, 20,000 × g), the pellet was once more extracted with 200 µL of cold methanol: chloroform: water (5:2:2). The two supernatants were combined and vacuum dried at 30 oC (Speedvac, Eppendorf Vacuum Concentrator Plus/5301). Dried extracts were reconstituted in methanol, centrifuged at 20,000 × g for 10 min, filtered through 0.22 µm PTPE membranes (Thermo Scientific, MA, USA) and filtrate collected in 1.5 mL LC/MS certified sample vials. |
| Processing Storage Conditions: | Room temperature |
| Extract Storage: | On ice |
| Sample Resuspension: | Methanol |
Combined analysis:
| Analysis ID | AN002288 | AN002289 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Waters Acquity H-Class | Waters Acquity H-Class |
| Column | Waters C18 (100 x 2.1mm,1.7um) | Waters C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Waters Synapt G2 XS QTOF | Waters Synapt G2 XS QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | abundance | abundance |
Chromatography:
| Chromatography ID: | CH001681 |
| Chromatography Summary: | MS analysis was conducted in an ultra-performance liquid chromatography (UPLC Acquity H-Class, Waters, Milford, USA) coupled to a quadrupole time of flight (QTof) G2-XS mass spectrometer (Waters, Milford, USA). The chromatographic separation was carried out in a C18 column (2.1×100 mm, 1.7 µm, Waters, Milford, USA), kept at 45 °C. The injection volume was 5 µL, and the flow rate set at 0.450 mL min−1. Mobile phases consisted of 0.1 % formic acid in Milli-Q water (A) and methanol (B). The gradient elution profile was as follow (time, % B): 0 min, 2% B; 0.25 min, 2 % B; 12.25 min, 99 % B; 13.0 min, 99 % B; 13.01 min; 2 % B; 17.00 min; 2 % B and then the column was equilibrated for 5 min prior to each analysis. |
| Instrument Name: | Waters Acquity H-Class |
| Column Name: | Waters C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Rate: | 0.450 mL min−1 |
| Sample Injection: | 5 |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Analytical Time: | 17 min |
| Time Program: | 17 min |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002132 |
| Analysis ID: | AN002288 |
| Instrument Name: | Waters Synapt G2 XS QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS analysis was conducted in an ultra-performance liquid chromatography (UPLC Acquity H-Class, Waters, Milford, USA) coupled to a quadrupole time of flight (QTof) G2-XS mass spectrometer (Waters, Milford, USA). The chromatographic separation was carried out in a C18 column (2.1×100 mm, 1.7 µm, Waters, Milford, USA), kept at 45 °C. The injection volume was 5 µL, and the flow rate set at 0.450 mL min−1. Mobile phases consisted of 0.1 % formic acid in Milli-Q water (A) and methanol (B). The gradient elution profile was as follow (time, % B): 0 min, 2% B; 0.25 min, 2 % B; 12.25 min, 99 % B; 13.0 min, 99 % B; 13.01 min; 2 % B; 17.00 min; 2 % B and then the column was equilibrated for 5 min prior to each analysis. The MS acquisition was performed in negative and positive ionization modes in a scan range from m/z 100 to 1200 and time acquisition of 0 to 17 min. The analysis type performed was accurate mass screening on MSE data with a low collision energy of 4.00 eV and a high-energy ramp of 10.00 to 45.00 eV. The capillary and cone voltage were set at 2.50 kV and 40 V, respectively. The desolvation gas was set to 600 L h−1, the cone gas set to 50 L h−1 and the source and desolvation temperature was set to 100 °C and 250 °C, respectively. For automated accurate mass measurement, a solution of leucine-enkephalin (200 ng mL−1) in methanol: water (50:50) with 0.1% formic acid was used as lock mass and pumped at a flow rate of 5 µL min−1. The molecule of leucine-enkephalin (m/z 556.2766 in ESI+ and m/z 554.262 in ESI−) was used for recalibrating the mass axis and ensuring a robust accurate mass measurement at any time. For continuous quality assurance and to provide confidence in the data, quality control (QC), a mix prepared from equal volumes of all the samples was injected between every three samples in the batch along with methanol as a blank run to correct a drift of the raw signal intensity during the analysis. All the data acquired were exported by Waters UNIFI software in order to analyze by the software Progenesis QI (Nonlinear Dynamics, Newcastle, United Kingdom). |
| Ion Mode: | POSITIVE |
| Collision Energy: | 4.00 eV |
| Fragment Voltage: | High-ernergy ramp of 10.00 to 45.00 eV |
| Fragmentation Method: | MSE |
| MS ID: | MS002133 |
| Analysis ID: | AN002289 |
| Instrument Name: | Waters Synapt G2 XS QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS analysis was conducted in an ultra-performance liquid chromatography (UPLC Acquity H-Class, Waters, Milford, USA) coupled to a quadrupole time of flight (QTof) G2-XS mass spectrometer (Waters, Milford, USA). The chromatographic separation was carried out in a C18 column (2.1×100 mm, 1.7 µm, Waters, Milford, USA), kept at 45 °C. The injection volume was 5 µL, and the flow rate set at 0.450 mL min−1. Mobile phases consisted of 0.1 % formic acid in Milli-Q water (A) and methanol (B). The gradient elution profile was as follow (time, % B): 0 min, 2% B; 0.25 min, 2 % B; 12.25 min, 99 % B; 13.0 min, 99 % B; 13.01 min; 2 % B; 17.00 min; 2 % B and then the column was equilibrated for 5 min prior to each analysis. The MS acquisition was performed in negative and positive ionization modes in a scan range from m/z 100 to 1200 and time acquisition of 0 to 17 min. The analysis type performed was accurate mass screening on MSE data with a low collision energy of 4.00 eV and a high-energy ramp of 10.00 to 45.00 eV. The capillary and cone voltage were set at 2.50 kV and 40 V, respectively. The desolvation gas was set to 600 L h−1, the cone gas set to 50 L h−1 and the source and desolvation temperature was set to 100 °C and 250 °C, respectively. For automated accurate mass measurement, a solution of leucine-enkephalin (200 ng mL−1) in methanol: water (50:50) with 0.1% formic acid was used as lock mass and pumped at a flow rate of 5 µL min−1. The molecule of leucine-enkephalin (m/z 556.2766 in ESI+ and m/z 554.262 in ESI−) was used for recalibrating the mass axis and ensuring a robust accurate mass measurement at any time. For continuous quality assurance and to provide confidence in the data, quality control (QC), a mix prepared from equal volumes of all the samples was injected between every three samples in the batch along with methanol as a blank run to correct a drift of the raw signal intensity during the analysis. All the data acquired were exported by Waters UNIFI software in order to analyze by the software Progenesis QI (Nonlinear Dynamics, Newcastle, United Kingdom). |
| Ion Mode: | NEGATIVE |
| Collision Energy: | 4.00 eV |
| Fragment Voltage: | High-ernergy ramp of 10.00 to 45.00 eV |
| Fragmentation Method: | MSE |
