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

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Current revision (10:32, 17 September 2008) (view source)
 
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=== Mass spectra ===
=== Mass spectra ===
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== Reference ==
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== Reference ==
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* 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.]
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* 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.]
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[[Category:Flow injection analysis]]
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*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.]
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[[Category:Triple quadrupole]]
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*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.]
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[[Category:Glycerophospholipids (GP)]]
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[[Category:Flow_injection_analysis]] [[Category:ESI]] [[Category:Triple_quadrupole]] [[Category:Glycerophosphoserines_(GP03)]]

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 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

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

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