Offline nanoESI Phospholipid Profiling - LC-MS/MS

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 Mass spectrometer 3 Data analysis and quantification 3.1 Data handling 3.2 Calibration and quantification 4 Method validation 4.1 Precision 4.2 Limit of detection 5 Sample data 5.1 Mass spectra 5.2 Chromatogram 6 Reference

Sample preparation

• liquid-liquid extraction according to Bligh & Dyer
• sample sovlent: Lipid extracts are diluted 1:100 in acetonitrile/2-propanol 5:2

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Lipid extracts	10µl	24:0PC, 24:0PE, 24:0PS	500pmol each

Instrumentation and method

Mass spectrometer

• Type: Thermo TSQ Quantum Ultra
• Ionization mode: nanoESI
• Ionization voltage: 1500V
• Capillary temperature: 220°C
• Capillary voltage: 35V
• Collision gas: Argon
• Collision gas pressure: 0.7mTorr
• MS/MS-conditions:
  • scan range: m/z 400-900
  • resolution: 0.7 peak width FWHH
  • scan modes

Analyte	Precursor of [m/z]	Neutral loss [MW]	Collision energy [eV]
PC	184		35
PE		141	25
PS		185	25

Data analysis and quantification

Data handling

• Spectra are smoothed and peak lists exported to Excel.

Calibration and quantification

• calibration type: relative quantification
• The peak list generated from every headgroup scan is compared with a list of theoretical m/z values for every phospholipid class. Following peak identification the internal standard is set to 100% and all other peaks are expressed as % relative to internal standards.

Method validation

Precision

Limit of detection

Sample data

Mass spectra

Chromatogram

Reference

• Brügger et al. Proc Natl Acad Sci. 1997; 94(6):2339-2344

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