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.
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 | ST001818 |
Study Title | Quantification of PIPs species in mouse embryonic fibroblasts (MEFs) during autophagosome formation. |
Study Summary | Using 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 Name | Kono |
First Name | Nozomu |
Address | Hongo 7-3-1,, Bunyo-ku, Tokyo, 113-0033, Japan |
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 |
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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. |