A liquid chromatography-tandem mass spectrometry method for measurement of N-modified phosphatidylethanolamines.
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 gamma-ketoaldehydes [gammaKAs] 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(3)NH(2))-mediated deacylation has previously been used in headgroup analysis of phosphatidylinositol phosphates. Therefore, we developed an accurate assay for NAPEs and gammaKA-PEs using CH(3)NH(2)-mediated deacylation and quantitation of the resulting glycerophospho-N-modified ethanolamines by liquid chromatography-tandem mass spectrometry.
Guo
Lilu
L
Division of Clinical Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
Amarnath
Venkataraman
V
Davies
Sean S
SS
D016718:Arachidonic Acid / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D006868:Hydrolysis; D008744:Methylamines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D008744:Methylamines; D010714:Phosphatidylethanolamines; D018129:Phosphatidylinositol Phosphates; D016718:Arachidonic Acid; C027451:methylamine