Phosphatidylethanolamine - ESI-MS/MS - Liebisch et al.

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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
+
|-
|-
-
! Cultured cells
+
! Material
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| 100µg protein || PE 28:0, PE 40:0 (diphytanoyl) || 500ng each
+
! Material used
 +
! Internal Standard(s)
 +
! Internal Standard(s) added
|-
|-
-
! Human plasma
+
! Cultured cells
-
| 20µl |PE 28:0, PE 40:0 (diphytanoyl) || 400ng each
+
| 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 ==
+
== Instrumentation and method ==
 +
=== Pump  ===
-
=== 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:
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* Flow gradient:  
+
 
-
{| border="1" class="wikitable" style="text-align:center;"
+
{| class="wikitable" style="text-align: center" border="1"
-
! Time [min]!! Flow [ml/min] !! % Solvent A !! % Solvent B
+
|-
 +
! Time [min]
 +
! Flow [ml/min]  
 +
!  % Solvent A  
 +
!  % Solvent B
|-
|-
! 0  
! 0  
-
| 0.05 || 100 ||0
+
| 0.05  
 +
| 100  
 +
| 0
|-
|-
! 0.1  
! 0.1  
-
| 0.03 || 100 ||0
+
| 0.03  
 +
| 100  
 +
| 0
|-
|-
! 1.1  
! 1.1  
-
| 0.2 || 100 ||0
+
| 0.2  
 +
| 100  
 +
| 0
|-
|-
! 1.3  
! 1.3  
-
| 0.05 || 100 ||0
+
| 0.05  
 +
| 100  
 +
| 0
|}
|}
-
=== Autosampler ===
+
=== Autosampler ===
-
* Type: CTC Pal
+
-
* Injection volume: 20µl
+
-
* Wash solvent: methanol/chloroform = 1/1
+
-
=== Mass spectrometer ===
+
*Type: CTC Pal
-
* Type: Triple quadrupole (Micromass, Quattro Ultima)
+
*Injection volume: 20µl
-
* Ionization mode: ESI positive
+
*Wash solvent: methanol/chloroform = 1/1
-
* 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 ==
+
=== Mass spectrometer  ===
-
=== Data handling ===
+
*Type: Triple quadrupole (Micromass, Quattro Ultima)
-
* combine spectra above half peak height
+
*Ionization mode: ESI positive
-
* smooth combined spectrum (if necessary)
+
*Ionization voltage: 3500 V
-
* centroid combined spectrum
+
*Source temperature: 300°C
-
* pick peak intensities
+
*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
-
=== Isotope correction ===
+
== Data analysis and quantification  ==
-
* 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 ===
+
=== 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)
-
! Species!! Cultured cells !! Human plasma
+
*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"
|-
|-
-
! PE 34:1
+
! Species
-
| 0 - 500 pmol || 0 - 250 pmol
+
! Cultured cells
 +
! Human plasma
|-
|-
-
! PE 36:2
+
! PE 34:1
-
| 0 - 500 pmol || 0 - 250 pmol
+
| 0 - 500 pmol  
 +
| 0 - 250 pmol
|-
|-
-
! PE 38:4
+
! PE 36:2
-
| 0 - 500 pmol || 0 - 250 pmol
+
| 0 - 500 pmol  
 +
| 0 - 250 pmol
|-
|-
-
! PE 40:6
+
! PE 38:4
-
| 0 - 500 pmol || 0 - 250 pmol
+
| 0 - 500 pmol
 +
| 0 - 250 pmol
 +
|-
 +
! PE 40:6  
 +
| 0 - 500 pmol  
 +
| 0 - 250 pmol
|}
|}
-
== Method validation ==
+
== Method validation ==
 +
 
 +
=== Precision  ===
-
=== Precision ===
+
== Sample data  ==
-
== Sample data ==
+
=== Mass spectra  ===
-
=== Mass spectra ===
+
== Reference  ==
-
== 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:Flow injection analysis]]
+
[[Category:Flow_injection_analysis]] [[Category:ESI]] [[Category:Triple_quadrupole]] [[Category:Glycerophosphoethanolamines_(GP02)]]
-
[[Category:Triple quadrupole]]
+
-
[[Category:Glycerophospholipids (GP)]]
+

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

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

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