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MB Sample ID: SA314742
| Local Sample ID: | Hypoxia_1_8_pos |
| Subject ID: | SU002993 |
| Subject Type: | Plant |
| Subject Species: | Lycopersicon esculentum |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002993 |
| Subject Type: | Plant |
| Subject Species: | Lycopersicon esculentum |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Hypoxia_1_8_pos | SA314742 | FL037467 | hypoxia | condition |
Collection:
| Collection ID: | CO002986 |
| Collection Summary: | Control or hypoxia-exposed tomato roots were collected, minced, and extracted. Subsequently, lipidome profile was determined by LC-HRMS/MS |
| Collection Protocol Filename: | experimental procedure.pdf |
| Sample Type: | Plant roots |
| Collection Frequency: | 48h after hypoxia applied |
| Storage Conditions: | -20℃ |
Treatment:
| Treatment ID: | TR003002 |
| Treatment Summary: | Solanum lycopersicum (L.) cv. Moneymaker was cultivated under greenhouse conditions (14 h light 18 °C / 10 h dark 22 °C). Tomato plants grew on quartz sand culture for four weeks with a nutrient solution containing 5 mM NO3- [18,19]. Tomato roots were waterlogged for 48 h to apply hypoxic conditions. |
| Treatment: | watterlogging |
| Treatment Doseduration: | 48h |
| Plant Light Period: | 14h |
| Plant Temp: | 18°C |
Sample Preparation:
| Sampleprep ID: | SP002999 |
| Sampleprep Summary: | Lipids from tomato roots were extracted with a modified procedure adapted from Shiva and colleagues [20]. In brief, 0.1 g of grinded roots (cooled on liquid nitrogen) were added to a glass reaction tube filled with isopropanol supplemented with 0.01 % BHT. The mixture was incubated for 15 min at 75 °C and cooled on ice afterward. To the cooled-down mixture, 0.5 volume of chloroform, 0.2 volume of water and 3 µL of EquiSPLASH Lipidomix were added. Lipids were extracted for 1 h on ice with occasional vortexing in between. Reaction tubes were centrifuged at 5000 x g to induce phase separation, and the organic phase was transferred to another glass reaction tube. To the remaining water phase, 1.33 volumes of a chloroform:methanol mixture (2:1, v/v) supplemented with 0.1 % BHT was added and incubated for 30 min on ice with occasional vortexing. Reaction tubes were again centrifuged at 5000 rpm, and the chloroform phase was transferred to the second glass vial. The chloroform:methanol extraction step was repeated with the remaining water phase 3 times. Afterward, 0.33 volumes of KCl (1 M) were added to the combined lipid extracts and vortexed. The upper water phase was removed, and 0.66 volumes of water were added and vortexed. The upper phase was again removed, and sodium sulfate was added to remove the remaining water. Finally, the lipid extract was dried under constant N2-flow with a TurboVap sample evaporator (Biotage, Sweden) and frozen at -80°C until MS analysis. |
| Processing Storage Conditions: | On ice |
| Extract Storage: | -80℃ |
| Sample Resuspension: | chloroform:methanol:isopropanol (1:2:4 v/v/v, supplemented with 5 mM ammonium formate) |
| Sample Derivatization: | none |
| Sample Spiking: | EquiSPLASH |
Combined analysis:
| Analysis ID | AN004719 | AN004720 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Thermo Accucore C30 (150 x 2.1mm,2.6um) | Thermo Accucore C30 (150 x 2.1mm,2.6um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | normalized relative abundance | normalized relative abundance (a.u.) |
Chromatography:
| Chromatography ID: | CH003553 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Accucore C30 (150 x 2.1mm,2.6um) |
| Column Temperature: | 50°C |
| Flow Gradient: | 30% - 99% B 30min |
| Flow Rate: | 350µl/min |
| Solvent A: | 60% acetonitrile/40% water;10 mM ammonium formate; 0.1 % formic acid |
| Solvent B: | 90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1 % formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004465 |
| Analysis ID: | AN004719 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS raw files were analysed with the software LipidSearch (version 4.2.27, Thermo Fisher) using the following parameters: retention time interval 0.01 min, m-score threshold 5, precursor tolerance 5 ppm, product tolerance 8 ppm, ID quality filter A (fatty acid chain and class identified completely) and B (class and some fatty acid chains identified). The resulting text files were processed with an in-house KNIME workflow and filtered for ppm error, peak quality and area score. The area of identical lipids with different ion adducts was summed up. The resulting excel file was processed in R (v 4.0.2) with manual inspection of raw areas (Figure S1). Lipids in the blanks or blank extracts were excluded from the data, if not at least 5-fold lower in intensity in blank controls. Lipid areas were normalized to the respective lipids in the EquiSplash standard, and median normalization was applied (Figure S2). Lipids present in less than 20% of samples were excluded from subsequent analysis of lipid species. For principal component analyses (PCA), lipid species identified in at least 50% of samples were considered, and the three independent replicates were batch-adjusted using the ComBat algorithm. Lipids were annotated on the molecular species level in accordance with the proposed nomenclature by the LIPID MAPS consortium since no information on the sn-position of acyl/alkyl constituents was available |
| Ion Mode: | POSITIVE |
| MS ID: | MS004466 |
| Analysis ID: | AN004720 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS raw files were analysed with the software LipidSearch (version 4.2.27, Thermo Fisher) using the following parameters: retention time interval 0.01 min, m-score threshold 5, precursor tolerance 5 ppm, product tolerance 8 ppm, ID quality filter A (fatty acid chain and class identified completely) and B (class and some fatty acid chains identified). The resulting text files were processed with an in-house KNIME workflow and filtered for ppm error, peak quality and area score. The area of identical lipids with different ion adducts was summed up. The resulting excel file was processed in R (v 4.0.2) with manual inspection of raw areas (Figure S1). Lipids in the blanks or blank extracts were excluded from the data, if not at least 5-fold lower in intensity in blank controls. Lipid areas were normalized to the respective lipids in the EquiSplash standard, and median normalization was applied (Figure S2). Lipids present in less than 20% of samples were excluded from subsequent analysis of lipid species. For principal component analyses (PCA), lipid species identified in at least 50% of samples were considered, and the three independent replicates were batch-adjusted using the ComBat algorithm. Lipids were annotated on the molecular species level in accordance with the proposed nomenclature by the LIPID MAPS consortium since no information on the sn-position of acyl/alkyl constituents was available |
| Ion Mode: | NEGATIVE |
