A liquid chromatography–tandem mass spectrometry method for measurement of N-modified phosphatidylethanolamines

Abstract

N-Acyl phosphatidylethanolamines (NAPEs) are synthesised in response to stress in a variety of organisms from bacteria to humans. More recently, nonenzymatic modification of the ethanolamine headgroup of phosphatidylethanolamine (PE) by various aldehydes, including levuglandins/isoketals (which are γ-ketoaldehydes [γKAs] derived from arachidonic acid), has also been demonstrated. The levels of these various N-modified PEs formed during stress and their biological significance remain to be fully characterized. Such studies require an accurate, facile, and cost-effective method for quantifying N-modified PEs. Previously, NAPE and some of the nonenzymatically N-modified PE species have been quantified by mass spectrometry after hydrolysis to their constituent N-acylethanolamine by enzymatic hydrolysis, most typically with Streptomyces chromofuscus phospholipase D. However, enzymatic hydrolysis is not cost-effective for routine analysis of a large number of samples, and hydrolytic efficiency may vary for different N-modified PEs, making quantitation more difficult. Therefore, we sought a robust and inexpensive chemical hydrolysis approach. Methylamine (CH 3NH 2)-mediated deacylation has previously been used in headgroup analysis of phosphatidylinositol phosphates. Therefore, we developed an accurate assay for NAPEs and γKA–PEs using CH 3NH 2-mediated deacylation and quantitation of the resulting glycerophospho-N-modified ethanolamines by liquid chromatography–tandem mass spectrometry.