Phosphatidylserine - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
Contents |
Sample preparation
- Lipid extraction according to Bligh and Dyer
- internal standards are added prior lipid extraction
- to avoid the loss of lipids, extraction was performed in glassware
- samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Cultured cells | 100µg protein | PS 28:0, PS 40:0 (diphytanoyl) | 750ng each |
Human plasma | 20µl | PS 28:0, PS 40:0 (diphytanoyl) | 300ng each |
Instrumentation and method
Pump
- Type: binary high pressure gradient (Agilent 1100)
- Mode: isocratic flow gradient
- Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
- Flow gradient:
Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
---|---|---|---|
0 | 0.05 | 100 | 0 |
0.1 | 0.03 | 100 | 0 |
1.1 | 0.2 | 100 | 0 |
1.3 | 0.05 | 100 | 0 |
Autosampler
- Type: CTC Pal
- Injection volume: 20µl
- Wash solvent: methanol/chloroform = 1/1
Mass spectrometer
- Type: Triple quadrupole (Micromass, Quattro Ultima)
- Ionization mode: ESI positive
- Ionization voltage: 3500 V
- Source temperature: 300°C
- Collision gas: Argon
- Collision gas pressure: 1.0 10-3 Torr
- Collision energy: 20 V
- MS/MS-mode: neutral loss scan of m/z 185.0
Data analysis and quantification
Data handling
- combine spectra above half peak height
- smooth combined spectrum (if necessary)
- centroid combined spectrum
- pick peak intensities
Isotope correction
- using Excel Macros correcting the peak intensities in a sequential algorithm starting from low mass species
- five isotope peaks including the monoisotopic were used
- the detailed algorithm is described in the appendix of Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
Species | Cultured cells | Human plasma |
---|---|---|
PS 34:1 | 0 - 500 pmol | 0 - 150 pmol |
PS 36:2 | 0 - 500 pmol | 0 - 150 pmol |
PS 38:4 | 0 - 500 pmol | 0 - 150 pmol |
PS 40:6 | 0 - 500 pmol | 0 - 150 pmol |
Method validation
Precision
Sample data
Mass spectra
Reference
- Modified method according to the principles described by Brügger et al. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2339-44.
- Analytical setup is described in Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.