Glycerophospholipid Profiling - LC-MS/MS
From LipidomicsWiki
Contents |
Sample preparation
- Lipid extracts according to Bligh and Dyer are spiked with internal standards, and get diluted 1:100 in acetonitrile/2-propanol 5:2, 1% ammonia acetate, 0.1% formic acid.
| Material | Material used | Internal Standard | Internal Standard added |
|---|---|---|---|
| Platelets | 500 Mio cells | 24:0 PC, 24:0 PE, 24:0 PS | 10uM each |
| Cell culture | 10 Mio cells | 24:0 PC, 24:0 PE, 24:0 PS | 10uM each |
| Tissue | 5-100mg | 24:0 PC, 24:0 PE, 24:0 PS | 10uM each |
Instrumentation and method
Pump
- Type: Thermo Accela
- Mode: Gradient
- Solvent A: Water with 1% ammonia acetate and 0.1% formic acid
- Solvent B: Acetonitrile/2-propanol 5:2 with 1% ammonia acetate and 0.1% formic acid
- Gradient:
| Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
|---|---|---|---|
| 0 | 0.25 | 65 | 35 |
| 4.0 | 0.25 | 30 | 70 |
| 20.0 | 0.25 | 0 | 100 |
| 30.0 | 0.25 | 0 | 100 |
Column
- Type: Thermo Hypersil GOLD C18, 100x1 mm, 1.9µm
- Temperature: 50°C
Autosampler
- Type: Accela
- Injection volume: 5ul
- Wash solvent: Solvent B
Mass spectrometer
- Type: Thermo TSQ Quantum Ultra
- Ionization mode: positive ESI
- Ionization voltage: 4500V
- Capillary temperature: 220°C
- HESI temperature: 300°C
- Capillary voltage: 35V
- Collision gas: Argon
- Collision gas pressure: 0.7mTorr
- MS/MS-conditions:
- MS/MS scan
| Analyte | Precursor [m/z] / Neutral loss | Scan range [m/z] | Collision energy [eV] |
|---|---|---|---|
| Phosphatidylcholines | 184 | 400 - 900 | 35 |
| Phosphatidylethanolamines | 141 | 400 - 900 | 25 |
| Phosphatidylserines | 185 | 400 - 900 | 22 |
| Dimethylphosphatidylethanolamines | 169 | 400 - 900 | 25 |
| Sphingomyelines | 184 | 400 - 900 | 35 |
Data analysis and quantitation
Data handling
- Peak areas are calculated by QuanBrowser. Identification is based on characteristic fragments and retention time.
Calibration and quantitation
- calibration type: relative quantitation
- The internal standard is set to 100% and all other peaks are expressed as % relative to internal standards.
