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MB Sample ID: SA316620
| Local Sample ID: | flg22(2-7) |
| Subject ID: | SU003030 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
| Age Or Age Range: | 4-5 weeks |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003030 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
| Age Or Age Range: | 4-5 weeks |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| flg22(2-7) | SA316620 | FL037712 | Col-0 | Genotype |
| flg22(2-7) | SA316620 | FL037712 | flg22 | Treatment |
Collection:
| Collection ID: | CO003023 |
| Collection Summary: | Apoplastic wash fluid (AWF) was isolated from the leaves of 5- to 6-week-old Arabidopsis plants treated with flg22 or a mock control. To initiate PAMP responses, a needle-less syringe was used to infiltrate the leaves with a solution of 100 nM flg22 in water, or with DMSO in water as a negative control. Six to eight leaves were infiltrated on each plant, and a total of six plants were infiltrated for each treatment condition. Individual plants were infiltrated with flg22 or the DMSO only control, never both. After eight hours, AWF was isolated by syringe-infiltrating the mock- and flg22-treated leaves with sterile H2O containing 164 µM ribitol as an internal standard. Immediately after infiltration, the aerial portion of the plant was removed by cutting the primary stem and briefly washed with H2O to remove surface contaminants. The infiltrated leaves were detached from the rosette and stacked between layers of parafilm, with 2 to 4 leaves in each layer. The parafilm booklet of leaves was wrapped with tape and suspended inside a 15 mL conical centrifuge tube. The tube was centrifuged at 750 x g for seven minutes. The AWF that collected at the bottom of the conical tube was transferred to a clean 1.7 mL microcentrifuge tube, then centrifuged at 21,000 x g for 10 minutes at 4°C. The resulting supernatant was transferred to a clean 1.7 mL microcentrifuge tube. After addition of 50 µL of chloroform, the samples were vortexed for 10 seconds and centrifuged at 21,000 x g for 10 minutes at 4°C. The upper aqueous phase was transferred to a clean 1.7 mL microcentrifuge tube, and the volume recovered was measured with a pipette. The AWF samples were then lyophilized to dryness and stored at -80°C until further use. |
| Sample Type: | Apoplastic washing fluid |
Treatment:
| Treatment ID: | TR003039 |
| Treatment Summary: | Plants were treated with 100 nM flg22 dissolved in H2O or a mock treatment for eight hours. |
Sample Preparation:
| Sampleprep ID: | SP003036 |
| Sampleprep Summary: | Samples were derivatized with 10 μL of 20 mg/mL methoxyamine hcl in pyridine for 90 minutes at 37 °C. Then 20 μL of MSTFA + 1% TMCS was added and the samples were incubated for 30 minutes at 37 °C. The samples were then randomly injected into the GC-MS. |
Combined analysis:
| Analysis ID | AN004787 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890B |
| Column | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 5977B |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH003618 |
| Chromatography Summary: | For each sample, a 20 µL aliquot of AWF was lyophilized to dryness and resuspended in 10 µL of 30 mg/mL methoxyamine hydrochloride (Sigma-Aldrich) in pyridine (Sigma-Aldrich). The resuspended sample was incubated at 37°C and shaken at 1800 rpm for 90 minutes. After adding 20 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (CovaChem), the sample was incubated at 37°C and shaken at 1800 rpm for 30 minutes. Each derivatized sample was injected with a 10:1 split into an Agilent 7890B GC system with a 30 m +10 m Duraguard x 0.25 mm x 0.25 μm DB-5MS+DG Agilent column. The oven temperature was kept at 60°C for 1 minute, then ramped to 300°C at a rate of 10°C/min and held at 300°C for 10 minutes. Analytes were detected with an Agilent 5977B MSD in EI mode scanning from 50 m/z to 600 m/z. Mass spectrum analysis, component identification and peak area quantification were performed with AMDIS (http://www.amdis.net/). To reduce erroneous identifications during automated feature identification, the FiehnLib library was used to create a custom library that included both identified compounds along with unidentified prominent peaks that were recorded as unknowns and designated by their respective retention times72. Statistics were performed with MetaboAnalyst73 and MetaboAnalystR74. An in house perl script was used to collate AMDIS-generated peak areas into an Excel spreadsheet. Peaks that were present in blank samples containing derivatization chemicals only were removed from the data set. Two to three technical replicates were analyzed for each sample and averaged to produce a single sample value. MetaboAnalyst settings were selected to replace missing values by 1/5 of the minimum positive value for each feature and normalize the peak areas of the features by the peak area of the internal ribitol standard in each respective sample. |
| Instrument Name: | Agilent 7890B |
| Column Name: | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| Column Temperature: | 60 ramped to 300 |
| Flow Gradient: | NA |
| Flow Rate: | .52 mL/min helium gas |
| Injection Temperature: | 250 |
| Internal Standard: | ribitol |
| Solvent A: | NA |
| Solvent B: | NA |
| Chromatography Type: | GC |
| Solvent C: | NA |
MS:
| MS ID: | MS004533 |
| Analysis ID: | AN004787 |
| Instrument Name: | Agilent 5977B |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | Data were collect with scans from 50 m/z to 600 m/z. Data files were processed with AMDIS using automated feature identification with a library adapted from FiehnLib mass spectral and retention index library. |
| Ion Mode: | POSITIVE |
