Characterization of Oxidized Phosphatidylethanolamine Derived from RAW 264.7 Cells Using 4-(Dimethylamino) Benzoic Acid Derivatives

Abstract

Recently, a derivative of PE, namely the 4-(dimethylamino)benzoic acid derivative has been developed with various isotope labeled variants that provided a universal precursor ion scan for diacyl, ether and plasmalogen PE lipids that can not be accomplished otherwise. This derivative was further investigated as a means to facilitate characterization of various oxidized phosphatidylethanolamine lipids by collision activation. Phospholipids derived from RAW 264.7 cells were treated with a free radical generating system to generate a complex mixture of oxidized and non-oxidized lipids that were separated by reversed-phase high-performance liquid chromatography and detected using a precursors of m/z 191 scan for the d₀-DMABA-labeled control sample and a precursor of m/z 197 scan for the d₆-DMABA-labeled oxidized sample. Collisional activation of the corresponding [M – H]⁻ ions permitted the identification of several chain shortened ω-aldehydes, as well as direct oxygen addition products including isoprostane PE and monohydroxy PE oxidized phospholipids that were not easily detected without the use of the DMABA derivatives. The stable isotope labeled derivatives permitted assessment of relative quantitative changes in oxidized lipids based upon ion abundance.