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MB Sample ID: SA354212
Local Sample ID: | 210309_MDA_Timecourse_RSL3_n2_24h_DMSO_22 |
Subject ID: | SU003381 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003381 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
210309_MDA_Timecourse_RSL3_n2_24h_DMSO_22 | SA354212 | FL041326 | Breast cancer cells | Sample source |
210309_MDA_Timecourse_RSL3_n2_24h_DMSO_22 | SA354212 | FL041326 | DMSO | Treatment |
210309_MDA_Timecourse_RSL3_n2_24h_DMSO_22 | SA354212 | FL041326 | 24 h | time point |
Collection:
Collection ID: | CO003374 |
Collection Summary: | Cultured cells were washed, trypsinized, counted and flash-frozen in liquid N2 and stored at -80°C. |
Sample Type: | Breast cancer cells |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR003390 |
Treatment Summary: | Treatment of brest cancer cells (MDA-MB-231 cells) for the analysis of oxidized phosphatidylethanolamine (oxPE), oxidized phosphatidylcholine (oxPC), and oxidized phosphatidylinositol (oxPI): Human MDA-MB-231 breast cancer cells were treated with vehicle (DMSO) or RSL3 (1 or 10 µM) with or without ferrostatin-1 (3 µM) for 2 h, 4 h, 6 h, or 24 h or with SCs (1, 3, and 10 µM) for 2 h at 37°C and 5% CO2. Cells were harvested, washed with PBS pH 7.4, snap-frozen, and stored at -80°C. |
Sample Preparation:
Sampleprep ID: | SP003388 |
Sampleprep Summary: | Phospholipids were extracted from cell pellets by successive addition of PBS pH 7.4, methanol, chloroform, and saline to a final ratio of 14:34:35:17. Evaporation of the organic layer yielded a lipid film that was dissolved in methanol and subjected to UPLC-MS/MS. |
Extract Storage: | -80℃ |
Combined analysis:
Analysis ID | AN005345 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Waters Acquity H-Class |
Column | Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) |
MS Type | ESI |
MS instrument type | QTRAP |
MS instrument name | ABI Sciex 6500+ |
Ion Mode | NEGATIVE |
Units | absolute intensities |
Chromatography:
Chromatography ID: | CH004047 |
Chromatography Summary: | Chromatographic separation of phospholipids was carried out on an Acquity BEH C8 column (1.7 μm, 130 Å, 2.1×100 mm, Waters, Milford, MA) using an ExionLC UHPLC system. |
Instrument Name: | Waters Acquity H-Class |
Column Name: | Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) |
Column Temperature: | 45°C |
Flow Gradient: | The gradient was ramped from 75 to 85% B over 5 min and further increased to 100% B within 2 min, followed by isocratic elution for another 2 min. |
Flow Rate: | 0.75 ml/min |
Solvent A: | water/acetonitrile 90/10, 2 mM ammonium acetate |
Solvent B: | water/acetonitrile 5/95, 2 mM ammonium acetate |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS005075 |
Analysis ID: | AN005345 |
Instrument Name: | ABI Sciex 6500+ |
Instrument Type: | QTRAP |
MS Type: | ESI |
MS Comments: | Targeted MRM with pre-optimized settings and subsequent automated integration of selected signals using Analyst 1.6.3 or Analyst 1.7.1 (Sciex). Oxidized phospholipid anions (PE and PI: [M-H]-; PC: [M+OAc]-) were detected after fragmentation to both fatty acid anions. Retention time windows for oxidized PC and oxidized PE were predicted based on the analysis of oxidized PC(16:0/20:4) (oxPAPC) (Avanti Polar Lipids, Alabaster, AL; 1[O]: 2.8–4.6 min; 2[O]: 2.9–4.3 min; 3[O]: 1.45–2.3 min). Quantitation is based on the most intense signals of oxidized fatty acid anion fragments. The fractions of phospholipids with one 1[O], two 2[O], or three oxygens 3[O] incorporated comprise multiple isomeric species that were summed and normalized to DMPC (for oxidized PC and oxidized PI) or DMPE (for oxidized PE) and cell number. |
Ion Mode: | NEGATIVE |