Summary of Study ST001816

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001149. The data can be accessed directly via it's Project DOI: 10.21228/M8611G This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST001816
Study Title	Quantification of PIPs species in mouse tissues.
Study Summary	Phosphoinositides (PIPs) species, differing in phosphorylation at hydroxyls of the inositol head group, play roles in various cellular events. Here we developed a supercritical fluid chromatography-mass spectrometry (SFC-MS) method that allows us to quantify molecular species of all seven PIP regioisomers in culture cells and tissues. Using this methodology, we quantified PIPs species in 13 mouse tissues.
Institute	Grad Sch of Pharmaceut Sci, Univ of Tokyo
Last Name	Kono
First Name	Nozomu
Address	Hongo 7-3-1,, Bunyo-ku, Tokyo, 113-0033, Japan
Email	nozomu@mol.f.u-tokyo.ac.jp
Phone	+81-3-5841-4723
Submit Date	2021-06-01
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2022-05-04
Release Version	1

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Project:

Project ID:	PR001149
Project DOI:	doi: 10.21228/M8611G
Project Title:	Quantification of PIPs species in biological samples.
Project Summary:	Phosphoinositides (PIPs) species, differing in phosphorylation at hydroxyls of the inositol head group, play roles in various cellular events. Here we developed a supercritical fluid chromatography-mass spectrometry (SFC-MS) method that allows us to quantify molecular species of all seven PIP regioisomers in culture cells and tissues.
Institute:	Grad Sch of Pharmaceut Sci, Univ of Tokyo
Last Name:	Kono
First Name:	Nozomu
Address:	Hongo 7-3-1,, Bunyo-ku, Tokyo, 113-0033, Japan
Email:	nozomu@mol.f.u-tokyo.ac.jp
Phone:	+81-3-5841-4723

Subject:

Subject ID:	SU001893
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	8 weeks old
Gender:	Male

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Tissue
SA168974	Brain3	Brain
SA168975	Brain2	Brain
SA168976	Brain1	Brain
SA168977	Colon3	Colon
SA168978	Colon1	Colon
SA168979	Colon2	Colon
SA168980	Heart3	Heart
SA168981	Heart1	Heart
SA168982	Heart2	Heart
SA168983	Kidney3	Kidney
SA168984	Kidney2	Kidney
SA168985	Kidney1	Kidney
SA168986	Liver2	Liver
SA168987	Liver1	Liver
SA168988	Liver3	Liver
SA168989	Lung2	Lung
SA168990	Lung1	Lung
SA168991	Lung3	Lung
SA168992	Pancreas2	Pancreas
SA168993	Pancreas1	Pancreas
SA168994	Pancreas3	Pancreas
SA168995	Skeletal Muscle1	Skeletal Muscle
SA168996	Skeletal Muscle2	Skeletal Muscle
SA168997	Skeletal Muscle3	Skeletal Muscle
SA168998	Small Intestine3	Small Intestine
SA168999	Small Intestine2	Small Intestine
SA169000	Small Intestine1	Small Intestine
SA169001	Spleen2	Spleen
SA169002	Spleen3	Spleen
SA169003	Spleen1	Spleen
SA169004	Stomach2	Stomach
SA169005	Stomach3	Stomach
SA169006	Stomach1	Stomach
SA169007	Testis1	Testis
SA169008	Testis2	Testis
SA169009	Testis3	Testis
SA169010	White Adipose Tissue3	White Adipose Tissue
SA169011	White Adipose Tissue2	White Adipose Tissue
SA169012	White Adipose Tissue1	White Adipose Tissue

Showing results 1 to 39 of 39

Showing results 1 to 39 of 39

Collection:

Collection ID:	CO001886
Collection Summary:	Wild-type male mice (C57BL/6J) were anaesthetized, perfused and tissues were harvested. Isolated mouse tissues were rinsed with cold PBS and immediately frozen in liquid nitrogen.
Sample Type:	Various Tissues

Treatment:

Treatment ID:	TR001906
Treatment Summary:	Thirteen tissues were harvested from 8-week-old wild-type male mice (C57BL/6J).

Sample Preparation:

Sampleprep ID:	SP001899
Sampleprep Summary:	The following solutions were prepared for lipid extractions; the quench mixture comprising 484 ml MeOH, 242 ml CHCl3, and 23.55 ml 1 M HCl; the pre-derivatization wash composed of 240 ml CHCl3, 120 ml MeOH and 90 ml 0.01 M HCl; and the post-derivatization wash made up of 240 ml CHCl3, 120 ml MeOH, and 90 ml H2O. Wash mixtures were shaken and allowed to separate into two phases. For pre/post-derivatization wash, the upper phase of each solution was used. Frozen tissues were ground into powder in liquid N2 using a pestle and mortar. Frozen tissues were collected into a safe-lock poly-propylene tube (2 ml), and resuspended with 750 μl of quench mix, 170 μl of H2O and internal standards [10 μl containing 2 ng 12:0/13:0 PI, 17:0/20:4 PI (4)P, 17:0/20:4 PI (4,5)P2 and 17:0/20:4 PI (3,4,5)P3], followed by vortex-mixing and lipid extraction. Lipid extraction was performed, based on procedures described by J Clark et al. The single-phase sample (a mixture of 170 μl of an aqueous sample, 2 ng of internal standards, and 750 μl of quench mix) were mixed with 725 μl of CHCl3 and 170 μl of 2M HCl, followed by vortex-mixing and centrifugation (15,000 g, 5 min at room temperature). The lower organic phase was collected into a fresh safe-lock poly-propylene tube (2 ml) and mixed with 708 μl of prederivatization wash, followed by vortex-mixing and centrifugation (15,000 g, 3 min at room temperature). The lower phase was collected into another fresh tube and subjected to derivatization. Derivatization of lipids was performed in a fume hood with adequate personal safety equipment as follows, based on procedures described by J Clark et al7. Fifty μl trimethylsilyl diazomethane in hexane (2 M solution; Sigma-Aldrich) was added to the lipid extracts (~1 ml), and after leaving to stand 10 min at room temperature, the reaction was quenched with 6 μl of acetic acid. Next, 700 μl post-derivatization wash solution was added to the mixture, followed by centrifugation (1,500 g, 3 min). The resultant lower phase was collected and rewashed with a 700 μl post-derivatization wash solution. Then 90 μl of MeOH and 10 μl of H2O were added to the final collected lower phase. The samples were dried up with a centrifugal evaporator. Finally, samples were dissolved in 80μl MeOH, sonicated briefly, and 20 μl H2O was added. To avoid degradation, the samples were stored at -80ºC until use.

Sampleprep ID:

SP001899

Sampleprep Summary:

The following solutions were prepared for lipid extractions; the quench mixture comprising 484 ml MeOH, 242 ml CHCl3, and 23.55 ml 1 M HCl; the pre-derivatization wash composed of 240 ml CHCl3, 120 ml MeOH and 90 ml 0.01 M HCl; and the post-derivatization wash made up of 240 ml CHCl3, 120 ml MeOH, and 90 ml H2O. Wash mixtures were shaken and allowed to separate into two phases. For pre/post-derivatization wash, the upper phase of each solution was used. Frozen tissues were ground into powder in liquid N2 using a pestle and mortar. Frozen tissues were collected into a safe-lock poly-propylene tube (2 ml), and resuspended with 750 μl of quench mix, 170 μl of H2O and internal standards [10 μl containing 2 ng 12:0/13:0 PI, 17:0/20:4 PI (4)P, 17:0/20:4 PI (4,5)P2 and 17:0/20:4 PI (3,4,5)P3], followed by vortex-mixing and lipid extraction. Lipid extraction was performed, based on procedures described by J Clark et al. The single-phase sample (a mixture of 170 μl of an aqueous sample, 2 ng of internal standards, and 750 μl of quench mix) were mixed with 725 μl of CHCl3 and 170 μl of 2M HCl, followed by vortex-mixing and centrifugation (15,000 g, 5 min at room temperature). The lower organic phase was collected into a fresh safe-lock poly-propylene tube (2 ml) and mixed with 708 μl of prederivatization wash, followed by vortex-mixing and centrifugation (15,000 g, 3 min at room temperature). The lower phase was collected into another fresh tube and subjected to derivatization. Derivatization of lipids was performed in a fume hood with adequate personal safety equipment as follows, based on procedures described by J Clark et al7. Fifty μl trimethylsilyl diazomethane in hexane (2 M solution; Sigma-Aldrich) was added to the lipid extracts (~1 ml), and after leaving to stand 10 min at room temperature, the reaction was quenched with 6 μl of acetic acid. Next, 700 μl post-derivatization wash solution was added to the mixture, followed by centrifugation (1,500 g, 3 min). The resultant lower phase was collected and rewashed with a 700 μl post-derivatization wash solution. Then 90 μl of MeOH and 10 μl of H2O were added to the final collected lower phase. The samples were dried up with a centrifugal evaporator. Finally, samples were dissolved in 80μl MeOH, sonicated briefly, and 20 μl H2O was added. To avoid degradation, the samples were stored at -80ºC until use.

Combined analysis:

Analysis ID	AN002948
Analysis type	MS
Chromatography type	Unspecified
Chromatography system	Shimadzu Nexera UC
Column	ULTRON AF-HILIC-CD (250 × 2.1 mm,5.0um) Shinwa Chemicalstries
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 4500 Qtrap
Ion Mode	POSITIVE
Units	pmol/mg tissue

Chromatography:

Chromatography ID:	CH002183
Instrument Name:	Shimadzu Nexera UC
Column Name:	ULTRON AF-HILIC-CD (250 × 2.1 mm,5.0um) Shinwa Chemicalstries
Column Temperature:	4℃
Flow Gradient:	0–16 min: 5% B→20% B; 16.01–18 min: 40% B; 18.01–22 min: 5% B
Flow Rate:	1.5 ml/min
Solvent A:	supercritical carbon dioxide (SCCO2)
Solvent B:	2.5% water/97.5% methanol; 0.1% formic acid
Chromatography Type:	Unspecified

MS:

MS ID:	MS002738
Analysis ID:	AN002948
Instrument Name:	ABI Sciex 4500 Qtrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The instrument parameters of QTRAP4500 for positive ion mode were as follows: curtain gas, 20 psi; ionspray voltage, 4500 V; temperature, 300 ºC; ion source gas 1, 18 psi; ion source gas 2, 20 psi. and quantification was performed using MultiQuant™ software (SCIEX).
Ion Mode:	POSITIVE