Glycerophospholipid Profiling - FT-MS

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Contents

Sample preparation

  • Lipid extracts according to Bligh and Dyer are spiked with internal standards, and get diluted 1:100 in acetonitrile/2-propanol 5:2, 1% ammonia acetate, 0.1% formic acid.
Material Material used Internal Standards Internal Standard added
Tissue extracts 5 - 100 mg tissue 24:0 PC, 24:0 PE, 24:0 PS  10uM each

Instrumentation and method

Pump

  • Type: Thermo Accela
  • Mode: Gradient
  • Solvent A: Water with 1% ammonia acetate and 0.1% formic acid
  • Solvent B: Acetonitrile/2-propanol 5:2 with 1% ammonia acetate and 0.1% formic acid
  • Gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.25 65  35
4.0 0.25 30  70 
20.0 0.25 100 
30.0 0.25 100 

Column

  • Type: Thermo Hypersil GOLD C18, 100x1 mm, 1.9µm
  • Temperature: 50°C

Autosampler

  • Type: Accela
  • Injection volume: 5ul
  • Wash solvent: Solvent B

Mass spectrometer

  • Type: Thermo LTQ-FT Ultra
  • Ionization mode: positive ESI
  • Ionization voltage: 4500V
  • Capillary temperature: 250°C
  • Tube Lens: 120V
  • Capillary voltage: 35V
  • Damping gas: Helium
  • Mass calibration: <2ppm external
  • MS-conditions:
Analyte Scan range [m/z]
Phosphatidylcholines  300 - 1000 
Phosphatidylethanolamines  300 - 1000 
Phosphatidylserines  300 - 1000 
Plasmalogens  300 - 1000 
Lysophosphatidylcholines  300 - 1000 
Lysophosphatidylethanolamines  300 - 1000 
Sphingomyelines  300 - 1000 

Data analysis and quantitation

Data handling

  • Peak areas for all analytes are calculated by QuanBrowser. Peak identification is based on exact mass (<2ppm) and retention time.

Calibration and quantitation

  • calibration type: relative quantitation 
  • The internal standard is set to 100% and all other peaks are expressed as % relative to internal standards.

Method validation

Precision

Method precision for 5 injections is better than 15%. Inter day retention time stability is better than 1%. 

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