New covalent modifications of phosphatidylethanolamine by alkanals: mass spectrometry based structural characterization and biological effects

Abstract

The pathophysiology of numerous human disorders, such as atherosclerosis, diabetes, obesity and Alzheimer's disease, is accompanied by increased production of reactive oxygen species (ROS). ROS can oxidatively damage nearly all biomolecules, including lipids, proteins and nucleic acids. In particular, (poly)unsaturated fatty acids within the phospholipid (PL) structure are easily oxidized by ROS to lipid peroxidation products (LPP) carrying reactive carbonyl groups. Carbonylated LPP are characterized by high in vivo toxicity due to their reactivity with nucleophilic substrates (Lys-, Cys-and His-residues in proteins or amino groups of phosphatidylethanolamines [PE]). Adducts of unsaturated LPP with PE amino groups have been reported before, whereas less is known about the reactivity of saturated alkanals – which are significantly increased in vivo under oxidative stress conditions – towards nucleophilic groups of PLs.Here, we present a study of new alkanal-dipalmitoyl-phosphatidylethanolamine (DPPE) adducts by MS-based approaches, using consecutive fragmentation (MSn) and multiple reaction monitoring techniques. At least eight different DPPE–hexanal adducts were identified, including Schiff base and amide adducts, six of which have not been reported before. The structures of these new compounds were determined by their fragmentation patterns using MSn experiments. The new PE-hexanal adducts contained dimeric and trimeric hexanal conjugates, including cyclic adducts. A new pyridine ring containing adduct of DPPE and hexanal was purified by HPLC, and its biological effects were investigated. Incubation of peripheral blood mononuclear cells and monocytes with modified DPPE did not result in increased production of TNF-α as one selected inflammation marker. However, incorporation of modified DPPE into 1,2-dipalmitoleoyl-sn-phosphatidylethanolamine multilamellar vesicles resulted in a negative shift of the transition temperature, indicating a possible role of alkanal-derived modifications in changes of membrane structure. © 2014 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons, Ltd.

Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress

“…One more reason for the high reactivity of short chain dicarbonyls could be the relatively high hydrophilicity. It was demonstrated that peptides as relatively hydrophilic substrates preferentially react with shorter carbon chain aldehydes whereas aminophospholipids, as more hydrophobic compounds, are modified with longer chain carbonylated analogues [99].…”

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

“…Notably, the use of different ion activation modes as well as different ionization sources is a powerful tool to elucidate metabolite and drug structures in complex chemical mixtures . Understanding the fragmentation behavior and the product ions of FA under different MS conditions can help elucidate the structure of FA analogs, as well as facilitate identification of new candidate molecules …”

Mass spectrometry strategies to unveil modified aminophospholipids of biological interest

“…The MS spectrum of the reaction products are shown in Figure , which clearly depicts the characteristic mass shifts of 120 Da, 138 Da, 156 Da, corresponding to cyclized Schiff base (a), Schiff base (b), and Michael adduct (c), respectively. More recently, Annibal et al () employed high‐resolution Orbitrap MS analysis to identify modified derivatives of 1,2‐dipalmitoyl‐ sn ‐3‐glycerophosphoethanolamine (DPPE) arising from its incubation with hexanal and other alkanals. The MS spectrum of the products of the reaction between DPPE and hexanal acquired in positive ion mode, shown in Figure A, shows new ions that have mass shifts corresponding to the sequential adductions of up to three hexanal molecules, resulting in a series of monomeric, dimeric, and trimeric covalent adducts.…”

“…The ion at m/z 692.52 corresponds to [M+H] + molecular ion of DPPE; the ion at m/z 774.60 corresponds to the Schiff base adduct (mass shift of 82 Da); the ion at m/z 856.68 corresponds to the dimeric adduct, arising from the covalent adduction of a second molecule of hexanal (mass shift of 164 Da), the ion at m/z 874.69 was identified as the hydrated form of this dimer; the ion at m/z 936.74 corresponds to the pyridinium ring adduct formed by covalent adduction of a third hexanal molecule, followed by cyclization and loss of one H 2 O molecule; the ion at at m/z 954.75 corresponds to the hydrated precursor of the pyridinium adduct; the ion at m/z 938.76 corresponds to the trimeric adduct. The mechanism of the consecutive covalent adductions of hexanal to DPPE is resumed in Panel B. Reprinted with permission from Annibal et al (), copyright 2014 [John Wiley & Sons].…”

“…The schematic representations of the fragmentations are also illustrated. Reprinted with permission from Annibal et al (), copyright 2014 [John Wiley & Sons].…”