Summary of Study ST001893
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 PR001192. The data can be accessed directly via it's Project DOI: 10.21228/M8MX3X This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001893 |
| Study Title | Involvement of Mieap in Cardiolipin metabolism (part I) |
| Study Summary | Quantitative assessment of total cardiolipin (CL) and comparison of CL species conducted with A549 (Ad-Mieap infected vs. non-infected) and LS174T cells (LS174T-cont vs. Mieap-KD). The A549 cells were harvested 24 hr after infection with Ad-Mieap and were compared with non-infected cells by mass spectrometric analysis. The LS174T-cont and Mieap-KD cells incubated under a normal condition were harvested and subjected to mass spectrometric analysis. |
| Institute | National Cancer Center Japan Research Institute |
| Last Name | Ikari |
| First Name | Naoki |
| Address | 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan |
| nikari@ncc.go.jp | |
| Phone | +81-3-3542-2511 |
| Submit Date | 2021-07-30 |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-08-09 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001192 |
| Project DOI: | doi: 10.21228/M8MX3X |
| Project Title: | Mass Spectrometric Data of Cardiolipin in Cells under Mieap Expression |
| Project Summary: | Mass spectrometric data of cardiolipin in A549 (Ad-Mieap infected vs. non-infected) and LS174T cells (LS174T-cont vs. Mieap-KD) |
| Institute: | National Cancer Center Japan Research Institute |
| Last Name: | Ikari |
| First Name: | Naoki |
| Address: | 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan |
| Email: | nikari@ncc.go.jp |
| Phone: | +81-3-3542-2511 |
Subject:
| Subject ID: | SU001971 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | American Type Culture Collection |
| Cell Strain Details: | A549, LS174T |
| Cell Counts: | 10 million |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype/Treatment |
|---|---|---|
| SA175982 | A549 Ad-Mieap 1 | Ad-Mieap infection |
| SA175983 | A549 Ad-Mieap 2 | Ad-Mieap infection |
| SA175984 | LS174T Control 2 | Control |
| SA175985 | LS174T Control 1 | Control |
| SA175986 | LS174T Mieap-KD 2 | Mieap-knockdown |
| SA175987 | LS174T Mieap-KD 1 | Mieap-knockdown |
| SA175988 | A549 Non-infection 1 | Non-infection |
| SA175989 | A549 Non-infection 2 | Non-infection |
| Showing results 1 to 8 of 8 |
Collection:
| Collection ID: | CO001964 |
| Collection Summary: | Cells were collected using trypsin-EDTA. The cells were snap-frozen in liquid nitrogen after cell count, and subsequently stored at -80°C until lipidomic analysis. |
| Sample Type: | Cultured cells |
| Volumeoramount Collected: | 10 million cells/tube |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001983 |
| Treatment Summary: | Infection of the A549 cell line was carried out by adding viral solution (Ad-Mieap) to A549 cell monolayers, and incubating at 37°C for 120 min with brief agitation every 20 min. This was followed by the addition of culture medium and the return of the infected cells to the 37°C incubator. We established a Mieap-KD cell line using LS174T. Mieap expression was inhibited in the cell line by retroviral expression of short-hairpin RNA (shRNA) against the Mieap sequence. We also established LS174T-cont cells using the retroviral vector with target sequence for EGFP. The LS174T-cont and Mieap-KD cells were incubated under normal condition. |
| Treatment Doseduration: | A549 cells: 24 h; LS174T-cont and Mieap-KD cells: none (incubated under normal condition) |
| Treatment Vehicle: | A549 cells: viral solution (Ad-Mieap); LS174T-cont and Mieap-KD cells: none (incubated under normal condition) |
| Cell Storage: | stored at -80°C |
Sample Preparation:
| Sampleprep ID: | SP001977 |
| Sampleprep Summary: | Total lipids were extracted from samples using the Bligh-Dyer method. An aliquot of the organic phase was added to an equal volume of methanol before being loaded onto a DEAE-cellulose column (Wako Chemical) pre-equilibrated with chloroform. After successive washes with chloroform/methanol (1:1, v/v), acidic phospholipids were eluted with chloroform/methanol/HCl/water (12:12:1:1, v/v), followed by evaporation to dryness to yield a residue was soluble in methanol. |
| Extraction Method: | the Bligh-Dyer method |
Combined analysis:
| Analysis ID | AN003074 |
|---|---|
| Chromatography ID | CH002274 |
| MS ID | MS002861 |
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | UltiMate 3000 (Thermo Fisher Scientific) |
| Column | Waters XBridge C18 (150 x 1.0mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | pmol/10,000,000 cells |
Chromatography:
| Chromatography ID: | CH002274 |
| Chromatography Summary: | An aliquot of the organic phase was added to an equal volume of methanol before being loaded onto a DEAE-cellulose column (Wako Chemical) pre-equilibrated with chloroform. After successive washes with chloroform/methanol (1:1, v/v), acidic phospholipids were eluted with chloroform/methanol/HCl/water (12:12:1:1, v/v), followed by evaporation to dryness to yield a residue was soluble in methanol. |
| Instrument Name: | UltiMate 3000 (Thermo Fisher Scientific) |
| Column Name: | Waters XBridge C18 (150 x 1.0mm,3.5um) |
| Column Temperature: | 40 |
| Flow Gradient: | ratios of 60%/40% (0 min), 40%/60% (1 min), 20%/80% (9 min), 5%/95% (11-30 min), 95%/5% (31-35 min) and 60%/40% (45 min) |
| Flow Rate: | 25ul/min |
| Solvent A: | 50% isopropanol/10% methanol/40% water; 5 mM ammonium formate;0.05% ammonium hydroxide |
| Solvent B: | 100% isopropanol; 5 mM ammonium formate; 0.05% ammonium hydroxide |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002861 |
| Analysis ID: | AN003074 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Analyses were performed on a LC/MS/MS system consisting of a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with an electrospray ionization source and an UltiMate 3000 system (Thermo Fisher Scientific). Lipid samples were separated on a Waters X-Bridge C18 (150 × 1.0 mm, 3.5μm) at 40°C using a solvent step-gradient as follows: mobile phase A (isopropanol/methanol/water (5:1:4, v/v/v) supplemented with 5 mM ammonium formate and 0.05% ammonium hydroxide (28% in water))/mobile phase B (isopropanol supplemented with 5 mM ammonium formate and 0.05% ammonium hydroxide (28% in water)) ratios of 60%/40% (0 min), 40%/60% (1 min), 20%/80% (9 min), 5%/95% (11-30 min), 95%/5% (31-35 min) and 60%/40% (45 min). Flow rate was 25 μL/min. Source and ion transfer parameters applied were as follows. Spray voltage was 3.0 kV. For negative ionization modes, the sheath gas and capillary temperatures were maintained at 60 and 320 °C, respectively.The Orbitrap mass analyzer was operated at a resolving power of 70,000 in full-scan mode (scan range: 200–1800 m/z; automatic gain control (AGC) target:3e6) and of 35,000 in the Top 20 data-dependent MS2 mode (stepped normalized collision energy: 20, 30 and 40; isolation window: 4.0 m/z; AGC target: 1e5). |
| Ion Mode: | NEGATIVE |
