Ceramide - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
(Difference between revisions)
Line 78: | Line 78: | ||
=== Calibration and quantification === | === Calibration and quantification === | ||
- | * calibration | + | * calibration type: matrix calibration - addition of naturally occurring species |
* both [M+H]+ and [M+H-H2O]+ are used | * both [M+H]+ and [M+H-H2O]+ are used | ||
* species used for calibration | * species used for calibration |
Revision as of 16:54, 6 June 2008
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 5 mM ammonium formate/chloroform (3/1 = v/v)
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Cultured cells | 100µg protein | Cer 14:0, Cer 17:0 | 50ng each |
Human plasma | 20µl | Cer 14:0, Cer 17:0 | 50ng each |
Instrumentation and method
Pump
- Type: binary high pressure gradient (Agilent 1100)
- Mode: isocratic flow gradient
- Solvent(s): 5 mM ammonium formate/chloroform (3/1 = v/v)
- 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:
- Source temperature:
- Collision gas: Argon
- Collision gas pressure:
- MS/MS-conditions:
- precursor ion scan of m/z 264.2
- multiple reaction monitoring table of species observed frequently
Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
---|---|---|---|
Cer 14:0 - H2O | 492.5 | 264.2 | x |
Cer 14:0 | 510.5 | 264.2 | x |
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
- correction of istopic overlap according to principles described for Phosphatidylcholine / Sphingomyelin determination - Liebisch et al.
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- both [M+H]+ and [M+H-H2O]+ are used
- species used for calibration
Species | Cultured cells | Human plasma |
---|---|---|
Cer 16:0 | 0-35pmol | 0-50pmol |
Cer 18:0 | 0-35pmol | 0-50pmol |
Cer 20:0 | 0-35pmol | 0-50pmol |
Cer 24:1 | 0-35pmol | 0-50pmol |
Cer 24:0 | 0-35pmol | 0-50pmol |