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MB Sample ID: SA347869
Local Sample ID: | WT-IP4-FigS2b |
Subject ID: | SU003309 |
Subject Type: | Plant |
Subject Species: | Arabidopsis thaliana |
Taxonomy ID: | 3702 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003309 |
Subject Type: | Plant |
Subject Species: | Arabidopsis thaliana |
Taxonomy ID: | 3702 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
WT-IP4-FigS2b | SA347869 | FL040845 | Plant Seed | Sample source |
WT-IP4-FigS2b | SA347869 | FL040845 | Control | Treatment |
Collection:
Collection ID: | CO003302 |
Collection Summary: | 1 g total seedlings grown on agar medium for 15 d or 0.1 g dry seeds were collected for InsP5/InsP6 detection; For InsP3/InsP4/InsP5/InsP6/InsP7/InsP8 detection in seeds and seedlings, 10 g of 12-day-old seedlings or 2.4 g of dry seeds were used for InsPs enrichment with 300 mg of TiO2 beads for each sample. |
Sample Type: | Plant |
Treatment:
Treatment ID: | TR003318 |
Treatment Summary: | No treatment, All seedlings are planted on 1/2 MS plates, and the seeds are healthy and dry. |
Sample Preparation:
Sampleprep ID: | SP003316 |
Sampleprep Summary: | The enrichment of InsPs with TiO2 beads and SDS-PAGE assay were performed as described by Wilson et al47. In brief, 1 g total seedlings grown on agar medium for 15 d or 0.1 g dry seeds were ground in liquid nitrogen, suspended in 5 ml of 1 M cold perchloric acid, and kept rotating for 15 min at 4 ℃. After centrifugation at 12,000 g for 10 min at 4 ℃, the supernatant was transferred into a new tube containing 30 mg of TiO2 beads (5 mm Titansphere; GL Sciences, Japan) and kept rotating at 4 ℃ for 20 - 30 min. After centrifugation at 5000 g for 10 min at 4 ℃, the beads were transferred into a new 1.5 ml tube and washed with pre-cold PA for 3-5 times, then eluted with 500 µl of 10 % ammonia solution. The eluate was freeze-dried at -50 ℃ and resuspended with 50 µl of 10 % ammonia solution, and the enriched InsPs was resolved in a 33 % polyacrylamide / Tris–borate–EDTA (TBE) gel and stained with toluidine blue. 10 µM synthetic InsP5 (myo-Inositol-1,3,4,5,6-pentaphosphate ammonium salt, Cayman) and InsP6 (Sichem) were used as the markers and standards. In order to verify the specific components in the eluate, the total eluate was firstly freeze-dried, then dissolved with 100 µl 80 % acetonitrile. |
Combined analysis:
Analysis ID | AN005238 |
---|---|
Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Agilent 1290 Infinity |
Column | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
MS Type | Other |
MS instrument type | HPLC-MS/MS |
MS instrument name | Agilent 5973 |
Ion Mode | POSITIVE |
Units | mg/g |
Chromatography:
Chromatography ID: | CH003965 |
Chromatography Summary: | InsPs were detected using Hydrophilic Interaction High Performance Liquid Chromatography-Tandem Mass Spectrometry on an Agilent 1290 infinity HPLC system coupled to an Agilent 6460 triple Quad LC-MS/MS using InfinityLab Poroshell 120 HILIC-Z (2.1 × 100) column (Agilent Technologies, USA). Nitrogen was used as the sheath gas and drying gas. The nebulizer pressure was set to 45 psi and the flow rate of drying gas was 5 L / min. The flow rate and temperature of the sheath gas were 11 L / min and 350℃, respectively. Chromatographic separation was carried out on a HPLC column (100 × 2.1 mm, 2.7 µm). The column temperature was 35℃. The mobile phases consisted of (A) ammonium acetate in distilled water (pH 10.0) and (B) Acetonitrile. The gradient program was as follows: 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, 90 % of B. The flow rate was set at 0.3 ml / min, and the injection volume was 10 μl. Mass spectrometric detection was completed by use of an electrospray ionization (ESI) source in negative ion multiple-reaction monitoring (MRM) mode. InsPs were identified based on comparison to known InsPs species. The mass spectrometry parameters corresponding to different InsPs show as below: InsP3 (MRM: 419 -> 321, 419 -> 337, Acquisition time is 6-7 min); InsP4 (MRM: 499 -> 401, 499 -> 417, Acquisition time is 6-7 min); InsP5 (MRM: 579 -> 480.9, 579 -> 382.8, Acquisition time is 6-7 min); InsP6 (MRM: 659 -> 560.8, 659 -> 577, Acquisition time is 6-7 min); InsP7 (MRM: 739 -> 575, 739 -> 657, Acquisition time is 5-6 min); InsP8 (MRM: 819 -> 737, 819 -> 655, Acquisition time is 4-5 min). According to the regression equation calculated from the standard sample, substitute the response value of the sample into the equation to convert the corresponding concentration. For InsP3/InsP4/InsP5/InsP6/InsP7/InsP8 detection in seeds and seedlings, 10 g of 12-day-old seedlings or 2.4 g of dry seeds were used for InsPs enrichment with 300 mg of TiO2 beads for each sample. The enriched substances were analyzed by HPLC-MS/MS. The purchased InsP3 (1,4,5-InsP3, MedChemExpress), InsP4 (1,3,4,5-InsP4, MedChemExpress), InsP5 and InsP6, InsP7 (5-InsP7) and InsP8 (1,5-InsP8) from Lei lab25 were used as standard samples for generating the calibration curves. |
Instrument Name: | Agilent 1290 Infinity |
Column Name: | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
Column Temperature: | 35℃ |
Flow Gradient: | 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, 90 % of B |
Flow Rate: | 0.3 mL/min |
Solvent A: | 100% Distilled Water; 10% ammonium acetate (pH 10.0) |
Solvent B: | 100% Acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS004971 |
Analysis ID: | AN005238 |
Instrument Name: | Agilent 5973 |
Instrument Type: | HPLC-MS/MS |
MS Type: | Other |
MS Comments: | InsPs were detected using Hydrophilic Interaction High Performance Liquid Chromatography-Tandem Mass Spectrometry on an Agilent 1290 infinity HPLC system coupled to an Agilent 6460 triple Quad LC-MS/MS using InfinityLab Poroshell 120 HILIC-Z (2.1 × 100) column (Agilent Technologies, USA). Nitrogen was used as the sheath gas and drying gas. The nebulizer pressure was set to 45 psi and the flow rate of drying gas was 5 L / min. The flow rate and temperature of the sheath gas were 11 L / min and 350℃, respectively. Chromatographic separation was carried out on a HPLC column (100 × 2.1 mm, 2.7 µm). The column temperature was 35℃. The mobile phases consisted of (A) ammonium acetate in distilled water (pH 10.0) and (B) Acetonitrile. The gradient program was as follows: 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, 90 % of B. The flow rate was set at 0.3 ml / min, and the injection volume was 10 μl. Mass spectrometric detection was completed by use of an electrospray ionization (ESI) source in negative ion multiple-reaction monitoring (MRM) mode. InsPs were identified based on comparison to known InsPs species. The mass spectrometry parameters corresponding to different InsPs show as below: InsP3 (MRM: 419 -> 321, 419 -> 337, Acquisition time is 6-7 min); InsP4 (MRM: 499 -> 401, 499 -> 417, Acquisition time is 6-7 min); InsP5 (MRM: 579 -> 480.9, 579 -> 382.8, Acquisition time is 6-7 min); InsP6 (MRM: 659 -> 560.8, 659 -> 577, Acquisition time is 6-7 min); InsP7 (MRM: 739 -> 575, 739 -> 657, Acquisition time is 5-6 min); InsP8 (MRM: 819 -> 737, 819 -> 655, Acquisition time is 4-5 min). According to the regression equation calculated from the standard sample, substitute the response value of the sample into the equation to convert the corresponding concentration. |
Ion Mode: | POSITIVE |