Phosphatidylethanolamine - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
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- | == Sample preparation == | + | == Sample preparation == |
- | * [[Lipid extraction according to Bligh and Dyer]] | + | *[[Lipid extraction according to Bligh and Dyer|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 |
- | * samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v) | + | *samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v) |
- | {| class="wikitable" style="text-align:center" | + | {| class="wikitable FCK__ShowTableBorders" style="text-align: center" |
- | + | ||
|- | |- | ||
- | ! | + | ! Material |
- | + | ! Material used | |
+ | ! Internal Standard(s) | ||
+ | ! Internal Standard(s) added | ||
|- | |- | ||
- | ! Human plasma | + | ! Cultured cells |
- | | 20µl | | + | | 100µg protein |
+ | | PE 28:0, PE 40:0 (diphytanoyl) | ||
+ | | 500ng each | ||
+ | |- | ||
+ | ! Human plasma | ||
+ | | 20µl | ||
+ | | PE 28:0, PE 40:0 (diphytanoyl) | ||
+ | | 400ng each | ||
|} | |} | ||
- | == Instrumentation and | + | == Instrumentation and method == |
+ | === Pump === | ||
- | + | *Type: binary high pressure gradient (Agilent 1100) | |
- | * Type: binary high pressure gradient (Agilent 1100) | + | *Mode: isocratic flow gradient |
- | * Mode: isocratic flow gradient | + | *Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v) |
- | * Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v) | + | *Flow gradient: |
- | * Flow gradient: | + | |
- | {| | + | {| class="wikitable" style="text-align: center" border="1" |
- | ! Time [min] | + | |- |
+ | ! Time [min] | ||
+ | ! Flow [ml/min] | ||
+ | ! % Solvent A | ||
+ | ! % Solvent B | ||
|- | |- | ||
! 0 | ! 0 | ||
- | | 0.05 | + | | 0.05 |
+ | | 100 | ||
+ | | 0 | ||
|- | |- | ||
! 0.1 | ! 0.1 | ||
- | | 0.03 | + | | 0.03 |
+ | | 100 | ||
+ | | 0 | ||
|- | |- | ||
! 1.1 | ! 1.1 | ||
- | | 0.2 | + | | 0.2 |
+ | | 100 | ||
+ | | 0 | ||
|- | |- | ||
! 1.3 | ! 1.3 | ||
- | | 0.05 | + | | 0.05 |
+ | | 100 | ||
+ | | 0 | ||
|} | |} | ||
- | === Autosampler === | + | === Autosampler === |
- | + | ||
- | + | ||
- | + | ||
- | + | *Type: CTC Pal | |
- | * Type: | + | *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 141.0 | ||
- | === | + | == Data analysis and quantification == |
- | + | ||
- | + | ||
- | + | ||
- | === Calibration and quantification === | + | === Data handling === |
- | * calibration type: matrix calibration - addition of naturally occurring species | + | |
- | * species used for calibration | + | *combine spectra above half peak height |
- | {| class="wikitable" style="text-align:center" | + | *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 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15522827 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 | ||
+ | |||
+ | {| class="wikitable FCK__ShowTableBorders" style="text-align: center" | ||
|- | |- | ||
- | ! | + | ! Species |
- | + | ! Cultured cells | |
+ | ! Human plasma | ||
|- | |- | ||
- | ! PE | + | ! PE 34:1 |
- | | 0 - 500 pmol | + | | 0 - 500 pmol |
+ | | 0 - 250 pmol | ||
|- | |- | ||
- | ! PE | + | ! PE 36:2 |
- | | 0 - 500 pmol | + | | 0 - 500 pmol |
+ | | 0 - 250 pmol | ||
|- | |- | ||
- | ! PE 40:6 | + | ! PE 38:4 |
- | | 0 - 500 pmol | + | | 0 - 500 pmol |
+ | | 0 - 250 pmol | ||
+ | |- | ||
+ | ! PE 40:6 | ||
+ | | 0 - 500 pmol | ||
+ | | 0 - 250 pmol | ||
|} | |} | ||
- | == Method validation == | + | == Method validation == |
+ | |||
+ | === Precision === | ||
- | == | + | == Sample data == |
- | == | + | === Mass spectra === |
- | == | + | == Reference == |
- | + | *Modified method according to the principles described by [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9122196 Brügger et al. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2339-44.] | |
- | * Modified method according to the principles described by [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9122196 Brügger et al. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2339-44.] | + | *Analytical setup is described in [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15522827 Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.] |
- | * Analytical setup is described in [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15522827 Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.] | + | |
- | [[Category: | + | [[Category:Flow_injection_analysis]] [[Category:ESI]] [[Category:Triple_quadrupole]] [[Category:Glycerophosphoethanolamines_(GP02)]] |
- | [[Category: | + | |
- | [[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 10 mM ammonium acetate/chloroform (3/1 = v/v)
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Cultured cells | 100µg protein | PE 28:0, PE 40:0 (diphytanoyl) | 500ng each |
Human plasma | 20µl | PE 28:0, PE 40:0 (diphytanoyl) | 400ng 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 141.0
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 |
---|---|---|
PE 34:1 | 0 - 500 pmol | 0 - 250 pmol |
PE 36:2 | 0 - 500 pmol | 0 - 250 pmol |
PE 38:4 | 0 - 500 pmol | 0 - 250 pmol |
PE 40:6 | 0 - 500 pmol | 0 - 250 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.