Phospholipid class-specific profiling by LC/MS (Utrecht protocol)

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== Data analysis and quantification  ==
== Data analysis and quantification  ==

Revision as of 14:13, 28 October 2008

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 (PI, PA, PG, PS) 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: phospholipid class dependent


MS scan modes and settings
class
scan type
scan speed
DP
CE
PC/SM
precursor m/z +184
300 amu/s


PtdEtn
neutral loss 141 (pos)
300 amu/s


PtdEtn
precursor m/z -196
300 amu/s


PtdIns
precursor m/z -241
300 amu/s


PtdGro
precursor m/z -153
300 amu/s


PtdSer
neutral loss 87 (neg)
300 amu/s




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

Contour plot showing PtdCho profiling. Note the separation of lyso-species (5-15 min) from diradyl species (20-50 min), as well as the separation of sn-1 and sn-2 lyso species (double peaks between 5 min and 15 min).

Contour plot of PtdCho profiling. Note the separation of lyso- and diradyl species. Also note the separation of sn-1 and sn-2 lyso species.

Separation of isobaric species

Multiple examples of isobaric species that are separated by liquid chromatography

Image:PC_LCMSdetail.jpg


Reference


Please add categories as below for:

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