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.

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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.

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Study IDST001817
Study TitleQuantification of PIPs species in LPIAT1 KO mouse embryonic fibroblasts (MEFs).
Study SummaryUsing a supercritical fluid chromatography-mass spectrometry (SFC-MS)-based methodology, we quantified phosphoinositides (PIPs) species in LPIAT1 KO mouse embryonic fibroblasts (MEFs).
Institute
Grad Sch of Pharmaceut Sci, Univ of Tokyo
Last NameKono
First NameNozomu
AddressHongo 7-3-1,, Bunyo-ku, Tokyo, 113-0033, Japan
Emailnozomu@mol.f.u-tokyo.ac.jp
Phone+81-3-5841-4723
Submit Date2021-06-02
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2022-05-04
Release Version1
Nozomu Kono Nozomu Kono
https://dx.doi.org/10.21228/M8611G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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

Factors:

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

mb_sample_id local_sample_id Tissue
SA169013LPIAT1 KO #1-3LPIAT1 KO #1
SA169014LPIAT1 KO #1-2LPIAT1 KO #1
SA169015LPIAT1 KO #1-4LPIAT1 KO #1
SA169016LPIAT1 KO #1-7LPIAT1 KO #1
SA169017LPIAT1 KO #1-8LPIAT1 KO #1
SA169018LPIAT1 KO #1-1LPIAT1 KO #1
SA169019LPIAT1 KO #1-6LPIAT1 KO #1
SA169020LPIAT1 KO #1-5LPIAT1 KO #1
SA169021LPIAT1 KO #2-4LPIAT1 KO #2
SA169022LPIAT1 KO #2-3LPIAT1 KO #2
SA169023LPIAT1 KO #2-5LPIAT1 KO #2
SA169024LPIAT1 KO #2-6LPIAT1 KO #2
SA169025LPIAT1 KO #2-8LPIAT1 KO #2
SA169026LPIAT1 KO #2-7LPIAT1 KO #2
SA169027LPIAT1 KO #2-2LPIAT1 KO #2
SA169028LPIAT1 KO #2-1LPIAT1 KO #2
SA169029WT6WT
SA169030WT7WT
SA169031WT2WT
SA169032WT1WT
SA169033WT5WT
SA169034WT3WT
SA169035WT4WT
SA169036WT8WT
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.

Combined analysis:

Analysis ID AN002949
Analysis type MS
Chromatography type Other
Chromatography system Shimadzu Nexera UC
Column ULTRON AF-HILIC-CD (250 mm × 2.1 mm, 5.0 µm; Shinwa Chemical Industries)
MS Type ESI
MS instrument type Ion trap
MS instrument name ABI SCIEX 4500 QTrap
Ion Mode POSITIVE
Units pmol/1000000 cells

Chromatography:

Chromatography ID:CH002184
Instrument Name:Shimadzu Nexera UC
Column Name:ULTRON AF-HILIC-CD (250 mm × 2.1 mm, 5.0 µm; Shinwa Chemical Industries)
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:water/methanol (2.5/97.5, v/v) containing 0.1% (v/v) formic acid
Chromatography Type:Other

MS:

MS ID:MS002739
Analysis ID:AN002949
Instrument Name:ABI SCIEX 4500 QTrap
Instrument Type:Ion trap
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
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