Phosphatidylethanolamine-based plasmalogens - ESI-MS/MS - Liebisch et al.

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 Pump 2.2 Autosampler 2.3 Mass spectrometer 3 Data analysis and quantification 3.1 Data handling 3.2 Isotope correction 3.3 Calibration and quantification 4 Method validation 4.1 Precision 5 Sample data 5.1 Mass spectra 6 Reference

Sample preparation

• Lipid extraction according to Bligh and Dyer
• internal standards are added prior lipid extraction
• to avoid the loss of lipids, extraction was performed in glassware
• samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Cultured cells	100µg protein	PE 28:0, PE 40:0 (diphytanoyl)	500ng each
Human plasma	20µl	PE 28:0, PE 40:0 (diphytanoyl)	400ng each

Instrumentation and method

Pump

• Type: binary high pressure gradient (Agilent 1100)
• Mode: isocratic flow gradient
• Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
• Flow gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0.05	100	0
0.1	0.03	100	0
1.1	0.2	100	0
1.3	0.05	100	0

Autosampler

• Type: CTC Pal
• Injection volume: 20µl
• Wash solvent: methanol/chloroform = 1/1

Mass spectrometer

• Type: Triple quadrupole (Micromass, Quattro Ultima)
• Ionization mode: ESI positive
• Ionization voltage: 3500 V
• Source temperature: 300°C
• Collision gas: Argon
• Collision gas pressure: 1.0 10-3 Torr
• Collision energy: 18 V
• MS/MS-mode:
  • PE p16:0/x: neutral loss scan of m/z 364.3
  • PE p18:1/x: neutral loss scan of m/z 390.3
  • PE p18:0/x: neutral loss scan of m/z 392.3

Data analysis and quantification

Data handling

• combine spectra above half peak height
• smooth combined spectrum (if necessary)
• centroid combined spectrum
• pick peak intensities

Isotope correction

• using Excel Macros correcting the peak intensities in a sequential algorithm starting from low mass species
• five isotope peaks including the monoisotopic were used
• the detailed algorithm is described in the appendix of Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.

Calibration and quantification

• calibration type: matrix calibration - addition of naturally occurring species
• species used for calibration

Species	Cultured cells	Human plasma
PE p16:0/20:4	0 - 500 pmol	0 - 250 pmol

Method validation

Precision

Sample data

Mass spectra

Reference

• Modified method according to the principles described by Zemski-Berry et al. J Am Soc Mass Spectrom. 2004 Oct;15(10):1499-508.
• Analytical setup is described in Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.