Phospholipid class-specific profiling by LC/MS (Utrecht protocol)
From LipidomicsWiki
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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
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).
Separation of isobaric species
Multiple examples of isobaric species that are separated by liquid chromatography
Reference
Please add categories as below for:
- analytical techniques applied
- lipid classes analyzed (see lipid class categories)