Cross-oxidation of angiotensin II by glycerophosphatidylcholine oxidation products

Peptide and protein lipoxidation is a deleterious process which has been related to several degenerative conditions. In the present study, the interaction of lipid secondary oxidation products with peptides was investigated by evaluating the modifications occurring to angiotensin II (Ang-II) in the presence of an oxidizing polyunsaturated glycerophospholipid (1-palmitoyl-2-arachidonoyl-glycerophosphatidylcholine, PAPC). PAPC oxidation was promoted by Fenton chemistry and the oxidation products were incubated with Ang-II. The reaction products were finally analysed by off-line nanospray high-performance liquid chromatography/matrix-assisted laser desorption/ionization tandem mass spectrometry (nano-HPLC/MALDI-TOF-MS/MS). Ang-II was found to form adducts with 26 different aldehydes, leading to 37 distinct reaction products. Modification of Ang-II occurred through reaction with reactive carbonyl species (RCS) originating from fatty acyl chain cleavage, while interactions with the oxidized phospholipid could not be detected. Adduction was observed to occur both by Michael and Schiff base mechanisms, most prevalently taking place at the peptide N-terminus or the arginine residue. Histidine modification could only be demonstrated to occur via Michael addition with two aldehydes: 4-hydroxy-2-nonenal (HNE) and 2-octenal. The highly reactive 4-oxo-2-nonenal (ONE) was shown to react preferentially with the arginine side chain, while malondialdehyde addition could only be confirmed at the N-terminus. Aspartic acid oxidative decarboxylation, amino acid side chain oxidation, multiple adduction or peptide cross-links could not be perceived. The inability to detect these reaction products is indicative of their low abundance or non-existence in competitive reaction conditions. The multiplicity of peptide modifications described emphasizes the complexity of lipoxidation, the effects of which are not possible to fully understand by the evaluation of independent reaction products.