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

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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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