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MB Sample ID: SA214033
| Local Sample ID: | 32_EA_2-pos |
| Subject ID: | SU002325 |
| Subject Type: | Plant |
| Subject Species: | Camelina Sativa |
| Age Or Age Range: | 10 days after fertilization |
| Species Group: | Developing seeds |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002325 |
| Subject Type: | Plant |
| Subject Species: | Camelina Sativa |
| Age Or Age Range: | 10 days after fertilization |
| Species Group: | Developing seeds |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 32_EA_2-pos | SA214033 | FL026002 | Embyo axis | Tissue type |
| 32_EA_2-pos | SA214033 | FL026002 | 32 | Time (hours) |
Collection:
| Collection ID: | CO002318 |
| Collection Summary: | Plant growth and culture conditions: Plants were grown in greenhouses with day/night temperature maintained at 22/20°C, 40-50% relative humidity, and 16h day/8h night photoperiod. Intact siliques during the seed filling growth stage (15 days after fertilization) were excised and placed in sterile media containing a modified Linsmaier and Skoog medium23,24 with Gamborg’s vitamins (Sigma) and 5 mM MES buffer adjusted to pH 5.8. Fifty mM [U-13C6]glucose was used as labeled substrate, and the composition of the remaining carbon and nitrogen sources represented maternal phloem composition to minimize metabolic perturbation and to maintain pseudo in vivo conditions as previously described25. Silique culturing was performed in a 96-well plate with 0.3 mL of medium and a single silique per well, under continuous light (250 µmol m-2 s-1). Tissue was collected and flash frozen immediately after each time point (2, 4, 8, 16 and 32h). Uncultured siliques excised from the maternal plant were used as unlabeled (0h) controls. Frozen tissue was sectioned, on top of dry ice, to excise embryo from the siliques and to separate cotyledons from the embryo axis. Cotyledon samples were extracted and analyzed for lipids in triplicates. |
| Collection Protocol Filename: | 13CLipids_CamelinaSeeds_Methods.docx |
| Sample Type: | Seeds |
| Collection Location: | Donald Danforth Plant Science Center |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002337 |
| Treatment Summary: | Plants were grown in greenhouses with day/night temperature maintained at 22/20°C, 40-50% relative humidity, and 16h day/8h night photoperiod. Intact siliques during the seed filling growth stage (15 days after fertilization) were excised and placed in sterile media containing a modified Linsmaier and Skoog medium23,24 with Gamborg’s vitamins (Sigma) and 5 mM MES buffer adjusted to pH 5.8. Fifty mM [U-13C6]glucose was used as labeled substrate, and the composition of the remaining carbon and nitrogen sources represented maternal phloem composition to minimize metabolic perturbation and to maintain pseudo in vivo conditions as previously described25. Silique culturing was performed in a 96-well plate with 0.3 mL of medium and a single silique per well, under continuous light (250 µmol m-2 s-1). Tissue was collected and flash frozen immediately after each time point (2, 4, 8, 16 and 32h). Uncultured siliques excised from the maternal plant were used as unlabeled (0h) controls. Frozen tissue was sectioned, on top of dry ice, to excise embryo from the siliques and to separate cotyledons from the embryo axis. Cotyledon samples were extracted and analyzed for lipids in triplicates. |
Sample Preparation:
| Sampleprep ID: | SP002331 |
| Sampleprep Summary: | Frozen cotyledon samples from Camelina were homogenized using a tissue lyser and the extraction of lipids was carried out using a phase separation method previously described26. Briefly, 1 mL 7:3 methanol:chloroform (-20°C) containing the ultimateSPLASHTM ONE lipid mix (Avanti Polar lipids, Alabaster, AL) as internal standard (1:20 dilution) was added to the samples, vortexed vigorously and incubated on a rotary shaker for 2 hours at 4°C. After incubation, 500 µL of ice-cold water was added to the samples, vortexed and centrifuged at 14,000 rpm at 4°C for 10 min to achieve phase separation. The upper aqueous phase was carefully removed, 200 µL of methanol was added to the remaining organic phase containing lipids and centrifuged at 14,000 rpm for 5 min to pellet the debris. The organic phase (supernatant) was transferred to a glass tube and dried using a speedvac centrifuge. Samples were re-suspended in 200 µL of 49:49:2 acetonitrile: methanol: chloroform, filtered using 0.2 µm PTFE microcentrifuge filters and transferred to a glass vial for RPLC-HRMS analysis. |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | methanol:chloroform:water |
Combined analysis:
| Analysis ID | AN003655 | AN003656 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Dionex UltiMate 3000 RSLCnano | Dionex UltiMate 3000 RSLCnano |
| Column | Custom C8 - Higgins Analytical | Custom C8 - Higgins Analytical |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Fusion Tribrid Orbitrap | Thermo Fusion Tribrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Intensity | Intensity |
Chromatography:
| Chromatography ID: | CH002709 |
| Chromatography Summary: | Separations for lipidomics were achieved using the loading pump of a Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) operating at a flow rate of 40 µL min-1 equipped with a custom-made C8 column (100 x 0.5 x 5 µm) from Higgins Analytical Inc. (Mountain view, CA) re-packed from a nucleodur C8 Gravity column (Macherey-Nagel, Allentown, PA). Mobile phases comprised of 1% 1 M ammonium acetate, 0.1 % acetic acid in water (A) and 1% 1 M ammonium acetate, 0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol (B). The following gradient modified from a previously described method27 to adapt to micro flow was used; 0-1 min at 55% B, 4 min at 75% B, 12 min at 89% B, 15 min at 99% B, 18 min at 99% B and 20 min at 55% B followed by equilibration up to 30 min. |
| Instrument Name: | Dionex UltiMate 3000 RSLCnano |
| Column Name: | Custom C8 - Higgins Analytical |
| Column Temperature: | 40 |
| Flow Rate: | 0.04 mL min-1 |
| Internal Standard: | Equisplash |
| Solvent A: | 100% water; 0.1% acetic acid; 10 mM ammonium acetate |
| Solvent B: | 70% acetonitrile/30% isopropanol; 0.1% acetic acid; 10 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003406 |
| Analysis ID: | AN003655 |
| Instrument Name: | Thermo Fusion Tribrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The eluent was sprayed on to the HESI source of an Orbitrap Fusion Lumos Tribrid MS, operated with sheath gas, 25 arbitrary units; auxiliary gas, 5 arbitrary units; ion transfer tube temperature, 300oC; vaporizer temperature, 100oC; and S-lens RF level, 60. The spray voltage was 4 kV in both positive and negative modes. Full MS data were collected for mass ranges 450-1200 m/z at 240,000 resolution from both positive and negative modes simultaneously, using polarity switch. The AGC target was set to “Standard” and the maximum IT was set to 100 ms. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003407 |
| Analysis ID: | AN003656 |
| Instrument Name: | Thermo Fusion Tribrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The eluent was sprayed on to the HESI source of an Orbitrap Fusion Lumos Tribrid MS, operated with sheath gas, 25 arbitrary units; auxiliary gas, 5 arbitrary units; ion transfer tube temperature, 300oC; vaporizer temperature, 100oC; and S-lens RF level, 60. The spray voltage was 4 kV in both positive and negative modes. Full MS data were collected for mass ranges 450-1200 m/z at 240,000 resolution from both positive and negative modes simultaneously, using polarity switch. The AGC target was set to “Standard” and the maximum IT was set to 100 ms. |
| Ion Mode: | NEGATIVE |
