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Journal of Lipid Research, Vol. 46, 1038-1046, May 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Analysis of cell membrane aminophospholipids as isotope-tagged derivatives

Karin A. Zemski Berry and Robert C. Murphy¹

Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, CO 80045

¹ To whom correspondence should be addressed. e-mail: robert.murphy{at}uchsc.edu

Glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) lipids were reacted with a multiplexed set of differentially isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The resulting derivatized aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions (m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectrometric response of N-methylpiperazine amide-tagged aminophospholipids was probed using glycerophosphoethanolamine and glycerophosphoserine lipid standards. The [M+H]⁺ of each tagged aminophospholipid shifted 144 Da, and during collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting aminophospholipids was useful for an unbiased analysis of plasmalogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the [M-H]^– ions.

The isotope-tagged reagents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu²⁺/H₂O₂ to illustrate the ability of these reagents to aid in the mass spectrometric identification of aminophospholipid changes that occur during biological stimuli.

Supplementary key words glycerophosphoethanolamine lipids • glycerophosphoserine lipids • electrospray • tandem mass spectrometry • lipid oxidation

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