Summary of Study ST001818

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 IDST001818
Study TitleQuantification of PIPs species in mouse embryonic fibroblasts (MEFs) during autophagosome formation.
Study SummaryUsing a supercritical fluid chromatography-mass spectrometry (SFC-MS)-based methodology, we quantified phosphoinositides (PIPs) species in mouse embryonic fibroblasts (MEFs) from WT or FIP200 KO mice during autophagosome formation.
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:SU001895
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Not applicable

Factors:

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

mb_sample_id local_sample_id Genotype Medium Bafilomycin
SA169040FIP200_KO_DMEM3FIP200_KO DMEM -
SA169041FIP200_KO_DMEM2FIP200_KO DMEM -
SA169042FIP200_KO_DMEM1FIP200_KO DMEM -
SA169037FIP200_KO_DMEM_Baf1FIP200_KO DMEM +
SA169038FIP200_KO_DMEM_Baf2FIP200_KO DMEM +
SA169039FIP200_KO_DMEM_Baf3FIP200_KO DMEM +
SA169046FIP200_KO_EBSS1FIP200_KO EBSS -
SA169047FIP200_KO_EBSS2FIP200_KO EBSS -
SA169048FIP200_KO_EBSS3FIP200_KO EBSS -
SA169043FIP200_KO_EBSS_Baf2FIP200_KO EBSS +
SA169044FIP200_KO_EBSS_Baf1FIP200_KO EBSS +
SA169045FIP200_KO_EBSS_Baf3FIP200_KO EBSS +
SA169052WT_DMEM2WT DMEM -
SA169053WT_DMEM1WT DMEM -
SA169054WT_DMEM3WT DMEM -
SA169049WT_DMEM_Baf1WT DMEM +
SA169050WT_DMEM_Baf2WT DMEM +
SA169051WT_DMEM_Baf3WT DMEM +
SA169058WT_EBSS2WT EBSS -
SA169059WT_EBSS3WT EBSS -
SA169060WT_EBSS1WT EBSS -
SA169055WT_EBSS_Baf1WT EBSS +
SA169056WT_EBSS_Baf2WT EBSS +
SA169057WT_EBSS_Baf3WT EBSS +
Showing results 1 to 24 of 24

Collection:

Collection ID:CO001888
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. For autophagy induction, 2 × 105 cells were seeded on 6-well plates and the next day, cells were washed twice with PBS and incubated in amino acid-free EBSS (starvation medium; Thermo Fisher Scientific 24010-043) in the presence or absence of bafilomycin A1 (Selleck Chemicals) for 30 min at 37 ºC. After the incubation, 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:TR001908
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. For autophagy induction, 2 × 105 cells were seeded on 6-well plates and the next day, cells were washed twice with PBS and incubated in amino acid-free EBSS (starvation medium; Thermo Fisher Scientific 24010-043) in the presence or absence of bafilomycin A1 (Selleck Chemicals) for 30 min at 37 ºC.

Sample Preparation:

Sampleprep ID:SP001901
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 AN002950
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 PI3P / 36:1PS

Chromatography:

Chromatography ID:CH002185
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:MS002740
Analysis ID:AN002950
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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