Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA347866

Local Sample ID:	WT-IP8-FigS2b
Subject ID:	SU003309
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702

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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-IP8-FigS2b	SA347866	FL040845	Plant Seed	Sample source
WT-IP8-FigS2b	SA347866	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.

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