SE18:/S2/M1
Sample Set Information
| ID | SE18 |
|---|---|
| Title | Global identification of phospholipid molecular species in mouse liver and brain by Orbitrap MS and automated search engine Lipid Search |
| Description | A comprehensive and precise identification of phospholipid molecular species was established with negative ion mode analyses by high accuracy MS and auto mated search engine "Lipid Search". Two tissues, liver (S01) and brain (S02) from mice were applied to the comprehensive analyses and differences in molecular species were detected. |
| Authors | Taguchi R and Ishikawa M; The University of Tokyo |
| Reference | Taguchi R and Ishikawa M (2010) Journal of Chromatography A 1217: 4229-4239 |
| Comment | The metadata is prepared by the Metabolonote administrator (Sakurai N). |
Sample Information
| ID | S2 |
|---|---|
| Title | Mouse brain |
| Organism - Scientific Name | Mus musculus |
| Organism - ID | NCBI taxonomy:10090 |
| Compound - ID | |
| Compound - Source | |
| Preparation | Male C57BL/6J mice (aged 2 months) were purchased from Japan SLC, Inc. (Shizuoka, Japan). Brain tissue was extracted and used. |
| Sample Preparation Details ID | |
| Comment |
Analytical Method Information
| ID | M1 |
|---|---|
| Title | LC-Orbitrap-MS, ESI Negative analysis |
| Method Details ID | MS1 |
| Sample Amount | 10.83 ug |
| Comment | The brain sample amount contained in the 1 ul solution injected to the reverse-phase liquid chromatography. |
Analytical Method Details Information
| ID | MS1 |
|---|---|
| Title | Preparation of lipid extract and ESI negative detection by LC-MS analysis |
| Instrument | LC-10ADVPuHPLC system (Shimadzu), LTQ Orbitrap (ThermoElectron) |
| Instrument Type | LC-Orbitrap-MS |
| Ionization | ESI |
| Ion Mode | Negative |
| Description | Preparation of total lipid extract
Liver (4.1 g) and brain (1.3 g) from three mice were homogenized with chloroform/methanol (1:2), and phospholipids were extracted by the Bligh and Dyer method. The total lipid extract was dried under a gentle stream of nitrogen, dissolved in 10 mL of chloroform/methanol (1:1) and stored at −20C. Acid hydrolysis of alkenylacyl species Direct acid hydrolysis of alkenylacyl species was performed using hydrochloric acid. Each 150 uL of 1M HCl and 150 uL of methanol were added to the 300 uL total lipid extract (chloroform / methanol / water, 1:2:1), and the mixed solution was agitated for 2 h. Then, 150 uL each of chloroform and H2O were added to the mixed solution. The solution was centrifuged at 3000 rpm for 5 min, and the lower organic solvent layer was obtained. Reverse-phase liquid chromatography Phospholipid molecular species were separated using reverse-phase liquid chromatography (RPLC) with a LC-10ADVPuHPLC system (Shimadzu, Kyoto, Japan) and Develosil C30 (150 mm×1.0 mm i.d., 3 um particles) column (Nomura Chemical Co. Ltd., Aichi, Japan). The 100 uL lipid extracts were re-dissolved in 100 uL methanol (liver; 300 uL) and 1 uL of sample was injected. Solvent A was acetonitrile–methanol–water (19:19:2) with 0.1% acetic acid and 0.1% ammonium hydroxide (28%), and solvent B was 2-propanol with 0.1% acetic acid and 0.1% ammonium hydroxide (28%). The initial mobile phase was 5% solvent B at a flow rate of 100 uL min−1. It was held for 5 min, and then linearly increased to 30% solvent B in 40 min, followed by isocratic elution at 50% solvent B for 41 to 90 min. The column was equilibrated at 5% solvent B for 15 min. ESI–MS analysis of phospholipids using LTQ Orbitrap High mass accuracy MS analysis and data-dependent MSn analysis using a LTQ Orbitrap hybrid mass spectrometer (ThermoElectron Corp., San Jose, CA) were applied to identify individual phospholipid molecular species. ESI–MS analysis was performed in the negative ion mode, MS spectra were obtained by the FT mode, and the data-dependent MS/MS (MS2) or MS3 spectra were obtained by IT mode. The instrument was calibrated externally with dipalmytoyl PC and palmytoyl lysoPC as standard every experimental day. MS spectra were acquired under operator control with target mass resolution of R = 100,000 at m/z 400. The ion spray voltage was set at −3500 V and the scan range of the instruments was set at m/z 200–1000. The trap fill-time was set at 200 ms. Nitrogen was used as curtain gas (set at 13 arbitrary units). Helium was used as a collision gas for CID experiments, and the collision energy was set at 30%. In negative ion mode, PE, PS, PI and PG are detected as [M−H]− ion, and choline containing phospholipids, such as PC and SM, are detected as [M+CH3COO]− ions. A polar head group specific neutral loss (choline-containing phosholipids; 74 Da, PS; 87 Da) from data-dependent MS/MS (MS2) combined with data-dependent MS3 was used for specific identification of PC, SM and PS. Triggering MS to MS2 or MS2 to MS3 was based on the ion intensity, by switching in a data-dependent manner. A precursor ion with the intensity higher than 100 counts was automatically selected as the product ion for the tandem mass spectra measurement both in MS2 and MS3. After two cycles of tandem mass spectra measurements, the ion exclusion was applied for 30 s, which means that this ion cannot be selected for tandem mass experiment during the exclusion time. |
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