Profiling of Phospholipids (Utrecht protocol)

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Contents

Sample preparation

  • Extraction according to Bligh and Dyer (with or without modifications to enhance recovery of acidic- and lyso-phospholipids and glyco(sphingo)lipids)
  • sample solvent: Samples stored dry under nitrogen atmosphere at -20oC

Instrumentation and method

Pump

  • Type: Perkin Elmer series 200 micropump (2x)
  • Mode: gradient and subsequent isocratic elution
  • Solvent(s): A: MeOH/Acetonitrile/H2O (45/30/25) B: MeOH/Acetonitrile (60/40). Both A and B contain 1 µM serine and 2.5 mM ammoniumacetate
  • Gradient (including re-equilibration for subsequent run):
  • Indication of operating pressure: around 120 bar
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.425 100 0
25 0.425 0 100
50
0.425 0 100
51
0.425
100
0
55
0.425
100
0


Column

  • Type: Synergi 4 µm MAX-RP 18A column (250 × 3 mm; Phenomenex, CA, US)
  • Temperature: ambient

Autosampler

  • Type: HTC PAL
  • Injection volume: 10 µl
  • Wash solvent: MeOH

Mass spectrometer

  • Type: 4000 QTRAP (Sciex)
  • Ionization mode: either negative (all classes) or positive (GPCho, GPEtn, SM)
  • Ionization voltage: -4500V or +4500V/5500V (optimize)
  • Source temperature(s): 450oC
  • Collision gas: nitrogen
  • Collision gas pressure: set to "medium"
  • Operating mode: Enhanced Mass Spectrometry (full scans using linear ion trap)


Data analysis and quantification

Data handling

  • Data are acquired with AnalystTM (version 1.4.2). Using Analyst's "Translat.exe", data may be converted to open formats such as mzXML and netCDF.

Isotope correction

  • Isotope correction is generally done by the data processing software (mzMine, XCMS).


Sample data

Contour Plot (detail)

Image:EMS_PL_profiling.jpg

Reference


Please add categories as below for:

  • analytical techniques applied
  • lipid classes analyzed (see lipid class categories)
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