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Originally published In Press as doi:10.1194/jlr.D300020-JLR200 on August 16, 2003

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Journal of Lipid Research, Vol. 44, 2181-2192, November 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology

Methods

Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation

Kim Ekroos1,*, Christer S. Ejsing1,*,{dagger}, Ute Bahr§, Michael Karas§, Kai Simons* and Andrej Shevchenko2,*

* Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
{dagger} The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark
§ Johann Wolfgang Goethe-University,, Marie-Curie-Strasse 9-11, 60439 Frankfurt am Main, Germany

2 To whom correspondence should be addressed. e-mail: shevchenko{at}mpi-cbg.de

The molecular composition of phosphatidylcholines (PCs) in total lipid extracts was characterized by a combination of multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer and MS3 fragmentation on an ion trap mass spectrometer. Precursor ion spectra for 50 acyl anion fragments of fatty acids (fatty acid scanning) acquired in parallel increased the specificity and the dynamic range of the detection of PCs and identified the fatty acid moieties in individual PC species. Subsequent analysis of detected PC peaks by MS3 fragmentation on an ion trap mass spectrometer quantified the relative amount of their positional isomers, thus providing the most detailed and comprehensive characterization of the molecular composition of the pool of PCs at the low-picomole level. The method is vastly simplified, compared with conventional approaches, and does not require preliminary separation of lipid classes or of individual molecular species, enzymatic digestion, or chemical derivatization. The approach was validated by the comparative analysis of the molecular composition of PCs from human red blood cells.

In the total lipid extract of Madin-Darby canine kidney II cells, we detected 46 PC species with unique fatty acid composition and demonstrated that the presence of positional isomers almost doubled the total number of individual molecular species.

Abbreviations: FAS, fatty acid scanning; lysoPC, lyso-phosphatidylcholine; M, mass of a zwitterionic PC; PC, phosphatidylcholine; PIS, precursor ion scanning; PIS m/z 184.1, scanning for precursor ions that produce a fragment ion with m/z 184.1 upon collision-induced dissociation; QqTOF, quadrupole time-of-flight; [R'CH2COO]-, acyl anion of a fatty acid with hydrocarbon moiety R'; X/Y-PC-PC, PC molecule with X fatty acid moiety at sn-1 position of the glycerol backbone, and Y fatty acid moiety at sn-2 position; {X; Y}-PC, PC molecule (or a mixture of isomeric molecules) comprising fatty acids X and Y at unidentified position of the backbone

Supplementary key words quadrupole time-of-flight • Madin-Darby canine kidney II cells • human red blood cells • mass spectrometry


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