Summary of Study ST001817

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	ST001817
Study Title	Quantification of PIPs species in LPIAT1 KO mouse embryonic fibroblasts (MEFs).
Study Summary	Using a supercritical fluid chromatography-mass spectrometry (SFC-MS)-based methodology, we quantified phosphoinositides (PIPs) species in LPIAT1 KO mouse embryonic fibroblasts (MEFs).
Institute	University 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	2021-06-02
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2022-05-04
Release Version	1

Select appropriate tab below to view additional metadata details:

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:	SU001894
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	8 weeks old
Gender:	Male
Species Group:	Mammals

Factors:

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

mb_sample_id	local_sample_id	Tissue
SA169013	LPIAT1 KO #1-3	LPIAT1 KO #1
SA169014	LPIAT1 KO #1-2	LPIAT1 KO #1
SA169015	LPIAT1 KO #1-4	LPIAT1 KO #1
SA169016	LPIAT1 KO #1-7	LPIAT1 KO #1
SA169017	LPIAT1 KO #1-8	LPIAT1 KO #1
SA169018	LPIAT1 KO #1-1	LPIAT1 KO #1
SA169019	LPIAT1 KO #1-6	LPIAT1 KO #1
SA169020	LPIAT1 KO #1-5	LPIAT1 KO #1
SA169021	LPIAT1 KO #2-4	LPIAT1 KO #2
SA169022	LPIAT1 KO #2-3	LPIAT1 KO #2
SA169023	LPIAT1 KO #2-5	LPIAT1 KO #2
SA169024	LPIAT1 KO #2-6	LPIAT1 KO #2
SA169025	LPIAT1 KO #2-8	LPIAT1 KO #2
SA169026	LPIAT1 KO #2-7	LPIAT1 KO #2
SA169027	LPIAT1 KO #2-2	LPIAT1 KO #2
SA169028	LPIAT1 KO #2-1	LPIAT1 KO #2
SA169029	WT6	WT
SA169030	WT7	WT
SA169031	WT2	WT
SA169032	WT1	WT
SA169033	WT5	WT
SA169034	WT3	WT
SA169035	WT4	WT
SA169036	WT8	WT

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO001887
Collection Summary:	MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. 1 × 106 cells were seeded on cell culture dishes (6.0 cm diameter) and the next day, cells were washed twice with PBS and collected to a safe-lock poly-propylene tube (2 ml) with 1 M HCl (500 μl), followed by centrifugation (15,000 g, 5 min at 4 ºC). Supernatants were removed rapidly and resuspended with 750 μl of quench mix, 170 μl of H2O.
Sample Type:	Fibroblasts

Treatment:

Treatment ID:	TR001907
Treatment Summary:	MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine.

Sample Preparation:

Sampleprep ID:	SP001900
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. Cells were washed twice with PBS and collected to a safe-lock poly-propylene tube (2 ml) with 1 M HCl (500 μl), followed by centrifugation (15,000 g, 5 min at 4 ºC). Supernatants were removed rapidly 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 extract steps. 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:

SP001900

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. Cells were washed twice with PBS and collected to a safe-lock poly-propylene tube (2 ml) with 1 M HCl (500 μl), followed by centrifugation (15,000 g, 5 min at 4 ºC). Supernatants were removed rapidly 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 extract steps. 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.

Chromatography:

Chromatography ID:	CH002184
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

Analysis:

Analysis ID:	AN002949
Analysis Type:	MS
Chromatography ID:	CH002184
Num Factors:	3
Num Metabolites:	138
Units:	pmol/1000000 cells