Ceramide - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
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== Sample preparation == | == Sample preparation == | ||
- | * | + | * [[Lipid extraction according to Bligh and Dyer]] |
* internal standards are added prior lipid extraction | * internal standards are added prior lipid extraction | ||
* to avoid the loss of lipids, extraction was performed in glassware | * to avoid the loss of lipids, extraction was performed in glassware | ||
Line 22: | Line 22: | ||
* Type: binary high pressure gradient (Agilent 1100) | * Type: binary high pressure gradient (Agilent 1100) | ||
* Mode: isocratic flow gradient | * Mode: isocratic flow gradient | ||
- | * Solvent(s): 5 mM ammonium formate/chloroform (3/1 = v/v) | + | * Solvent(s): Methanol containing 5 mM ammonium formate/chloroform (3/1 = v/v) |
- | * | + | * Flow gradient: |
{| border="1" class="wikitable" style="text-align:center;" | {| border="1" class="wikitable" style="text-align:center;" | ||
! Time [min]!! Flow [ml/min] !! % Solvent A !! % Solvent B | ! Time [min]!! Flow [ml/min] !! % Solvent A !! % Solvent B | ||
Line 48: | Line 48: | ||
* Type: Triple quadrupole (Micromass, Quattro Ultima) | * Type: Triple quadrupole (Micromass, Quattro Ultima) | ||
* Ionization mode: ESI positive | * Ionization mode: ESI positive | ||
- | * Ionization voltage: | + | * Ionization voltage: 3500 V |
- | * Source temperature: | + | * Source temperature: 250°C |
* Collision gas: Argon | * Collision gas: Argon | ||
- | * Collision gas pressure: | + | * Collision gas pressure: 1.0 10-3 Torr |
* MS/MS-conditions: | * MS/MS-conditions: | ||
** precursor ion scan of m/z 264.2 | ** precursor ion scan of m/z 264.2 | ||
Line 58: | Line 58: | ||
! Analyte !! Precursor [m/z] !! Precursor [m/z] !! Collision energy [eV] | ! Analyte !! Precursor [m/z] !! Precursor [m/z] !! Collision energy [eV] | ||
|- | |- | ||
- | ! Cer 14:0 - H2O | + | ! Cer 14:0 - H2O (IS) |
- | | 492.5|| 264.2 || | + | | 492.5|| 264.2 ||25 V |
|- | |- | ||
- | ! Cer 14:0 | + | ! Cer 14:0 (IS) |
- | | 510.5|| 264.2 || | + | | 510.5|| 264.2 ||25 V |
+ | |- | ||
+ | ! Cer 16:0 - H2O | ||
+ | | 520.5|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 16:0 | ||
+ | | 538.5|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 17:0 - H2O (IS) | ||
+ | | 534.5|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 17:0 (IS) | ||
+ | | 552.5|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 18:0 - H2O | ||
+ | | 548.5|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 18:0 | ||
+ | | 566.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 20:0 - H2O | ||
+ | | 576.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 20:0 | ||
+ | | 594.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 22:1 - H2O | ||
+ | | 602.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 22:1 | ||
+ | | 620.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 22:0 - H2O | ||
+ | | 604.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 22:0 | ||
+ | | 622.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 23:0 - H2O | ||
+ | | 618.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 23:0 | ||
+ | | 636.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 24:1 - H2O | ||
+ | | 630.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 24:1 | ||
+ | | 648.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 24:0 - H2O | ||
+ | | 632.6|| 264.2 ||25 V | ||
+ | |- | ||
+ | ! Cer 24:0 | ||
+ | | 650.6|| 264.2 ||25 V | ||
|- | |- | ||
|} | |} | ||
Line 75: | Line 129: | ||
=== Isotope correction === | === Isotope correction === | ||
- | * correction of istopic overlap according to principles described for [[ | + | * correction of istopic overlap according to principles described for [[Phosphatidylcholine / Sphingomyelin determination - Liebisch et al.]] |
=== 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 | ||
{| class="wikitable" style="text-align:center" | {| class="wikitable" style="text-align:center" | ||
- | ! Species | + | ! Species!! Cultured cells !! Human plasma |
|- | |- | ||
! Cer 16:0 | ! Cer 16:0 | ||
Line 88: | Line 142: | ||
|- | |- | ||
! Cer 18:0 | ! 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 | | 0-35pmol || 0-50pmol | ||
|} | |} | ||
- | |||
== Method validation == | == Method validation == | ||
- | |||
- | |||
=== Precision === | === Precision === | ||
+ | * CV over all: below 10% in plasma samples | ||
=== LOD === | === LOD === | ||
- | + | * 0.3 pmol injected amount (in fibroblast lipid extract) | |
- | + | ||
=== Recovery === | === Recovery === | ||
+ | * recovery of Cer 6:0 above 90% | ||
== Sample data == | == Sample data == | ||
Line 108: | Line 169: | ||
=== Mass spectra === | === Mass spectra === | ||
- | == | + | == Reference == |
- | + | *[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10428992 G.Liebisch et al. Journal of Lipid Research 1999, 40, 1539-1546.] | |
- | * [http://www. | + | |
- | [[Category: | + | [[Category:Flow_injection_analysis]] [[Category:ESI]][[Category:Triple_quadrupole]] [[Category:Ceramides_(SP02)]] |
- | [[Category: | + |
Current revision
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): Methanol containing 5 mM ammonium formate/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: 250°C
- Collision gas: Argon
- Collision gas pressure: 1.0 10-3 Torr
- 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 (IS) | 492.5 | 264.2 | 25 V |
Cer 14:0 (IS) | 510.5 | 264.2 | 25 V |
Cer 16:0 - H2O | 520.5 | 264.2 | 25 V |
Cer 16:0 | 538.5 | 264.2 | 25 V |
Cer 17:0 - H2O (IS) | 534.5 | 264.2 | 25 V |
Cer 17:0 (IS) | 552.5 | 264.2 | 25 V |
Cer 18:0 - H2O | 548.5 | 264.2 | 25 V |
Cer 18:0 | 566.6 | 264.2 | 25 V |
Cer 20:0 - H2O | 576.6 | 264.2 | 25 V |
Cer 20:0 | 594.6 | 264.2 | 25 V |
Cer 22:1 - H2O | 602.6 | 264.2 | 25 V |
Cer 22:1 | 620.6 | 264.2 | 25 V |
Cer 22:0 - H2O | 604.6 | 264.2 | 25 V |
Cer 22:0 | 622.6 | 264.2 | 25 V |
Cer 23:0 - H2O | 618.6 | 264.2 | 25 V |
Cer 23:0 | 636.6 | 264.2 | 25 V |
Cer 24:1 - H2O | 630.6 | 264.2 | 25 V |
Cer 24:1 | 648.6 | 264.2 | 25 V |
Cer 24:0 - H2O | 632.6 | 264.2 | 25 V |
Cer 24:0 | 650.6 | 264.2 | 25 V |
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 |
Method validation
Precision
- CV over all: below 10% in plasma samples
LOD
- 0.3 pmol injected amount (in fibroblast lipid extract)
Recovery
- recovery of Cer 6:0 above 90%