Characterization of Chain-Shortened Oxidized Glycerophosphocholine Lipids Using Fast Atom Bombardment and Tandem Mass Spectrometry

Abstract

Oxidized glycerophosphocholine (GPC) lipids produced by free-radical-initiated lipid peroxidation have been reported to activate human polymorphonuclear leukocytes exerting an effect through the platelet-activating factor receptor, yet few methods have been developed to characterize intact oxidized phospholipids, which may be present in small quantities. In this paper we detail the fast atom bombardment and collision-induced dissociation behavior of 1-hexadecanoyl-2-(5-oxopentanoyl)-GPC and 1-hexadecanoyl-2-pentanedioyl-GPC, two prototypic oxidized glycerophosphocholine lipids produced by hydroxyl radical-initiated peroxidation of 1-hexadecanoyl-2-arachidonoyl-GPC. Identities of these two products were established first using EI-GC/MS to analyze the corresponding diglyceride tert-butyldimethylsilyl ether derivatives. The aldehyde moiety of 1-hexadecanoyl-2-(5-oxopentanoyl)-GPC was found to react with FAB amine-containing liquid matrices (e.g., diethanolamine) to form an oxazolidine derivative (87 u increase in mass). No reaction was observed with matrices such as glycerol, thioglycerol, and 3-nitrobenzylalcohol. This derivatization can be used to distinguish an aldehyde-containing oxidized phospholipid from isobaric species not containing an aldehyde moiety. Results also showed that 1-hexadecanoyl-2-pentanedioyl-GPC formed primarily [M-H]− ions rather than the [M-15], [M-60], and [M-86] negative ions expected for GPC lipid species. The ionized ω-carboxyl group of the sn-2 substituent allowed the phosphocholine moiety to remain as a zwitterion. Following collision-induced dissociation, a N-methyl group from the choline was transferred to the ω-carboxyl of the sn-2 substituent prior to generation of the carboxylate anions, which resulted in a 14-u increase in the observed sn-2 carboxylate anion. This methyl transfer mechanism was confirmed through the use of 1-hexadecanoyl-2-nonanedioyl-[2H9]-GPC in which the carboxylate anion m/z was increased by 17 u following intramolecular transfer of a trideuterio methyl group from [2H9]choline.