Stable Isotope Labeled 4-(Dimethylamino)benzoic Acid Derivatives of Glycerophosphoethanolamine Lipids

Abstract

A set of four (D(0), D(4), D(6), and D(10)) deuterium enriched 4-(dimethylamino)benzoic acid (DMABA) N-hydroxysuccinimide (NHS) ester reagents was developed that react with the primary amine group of glycerophosphoethanolamine (PE) lipids to create derivatives where all subclasses of DMABA labeled PE are detected by a common precursor ion scan. The positive ion collision induced dissociation data from (D(0), D(4), D(6), and D(10))-DMABA labeled PE standards indicated that a precursor ion scan of m/z 191.1, 195.1, 197.1, and 201.1 could be used to selectively detect (D(0), D(4), D(6), and D(10))-DMABA modified PE, respectively, in a complex biological mixture. The PE lipids from a time course (0, 30, 60, and 300 min) of 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH) treatment of liposomes made of RAW 264.7 cell phospholipids were each labeled with the D(0)-, D(4)-, D(10)-, and D(6)-DMABA NHS ester reagents, respectively. The DMABA derivatives revealed loss of endogenous PE lipids and an increase in oxidized PE lipid throughout the time course of AAPH treatment. These DMABA NHS ester reagents provide a universal scan for diacyl, ether, and plasmalogen PE lipids that cannot be readily observed otherwise, enable differential labeling, and provide an internal standard for each PE lipid.