Peptide-phospholipid cross-linking reactions: Identification of leucine enkephalin-alka(e)nal-glycerophosphatidylcholine adducts by tandem mass spectrometry

Abstract

The covalent interactions between peptides and lipid oxidation products, with formation of Schiff and Michael adducts, are known to occur during free radical oxidative damage. In this study, leucine-enkephalin-glycerophosphatidylcholine alka(e)nal adducts were analyzed by electrospray tandem mass spectrometry (MS/MS). Upon collision-induced dissociation of the Leucine enkephalin-2-(9-oxo-nonanoyl)-1-palmitoyl-3-glycerophosphatidylcholine, an alkanal Schiff adduct observed at m/z 1187.7, the main product ions were attributed to the phosphocholine polar head and loss of the peptide. Also, product ions resulting from characteristic losses of phosphatidylcholines and cleavages of the peptide chain (mainly b-type) were observed. Additional product ions formed by combined peptide and phosphatidylcholine fragmentations were identified. The fragmentation pattern of the leucine enkephalin-alkanal Schiff adduct and the leucine enkephalin-alkenal phosphatidylcholine Schiff and Michael adducts were similar, although the loss of the peptide for the Michael adduct should occur through a distinct mechanism. These fragmentation pathways differ greatly from those described for peptide-lipid Schiff and Michael adducts, in which only peptide chain cleavages are reported, probably due to charge retention in the glycerophosphatidylcholine polar head in peptideglycerophosphatidylcholine adducts. O xidative damage of peptides/proteins has received increasing attention due to the growing evidence of being associated with diabetes, cancer, and several age-related diseases [1]. The damage can be induced by the structural modification of the peptide/protein by reactive oxygen species (ROS) and other radicalar species, or by cross-linking reactions between peptide/protein and the oxidation products of other biomolecules (lipids, phospholipids, and DNA bases) [1]. The peptide-lipid adducts occur by reaction of the primary amine group (1) with the terminal carbonyl group (named as Schiff adduct), or (2) with the double-bond present in unsaturated aldehydes (named as Michael adduct). Currently, the work published on the peptide-lipid covalent interactions has focused on the identification of peptide adducts formed with the 4-hydroxy-nonenal (4-HNE) [2], which is a secondary oxidation product of -6 lipids (such as linoleic and arachidonic acids) found in membrane phospholipids, in triglycerides, and in low density lipoprotein [3]. The studies published allowed to propose that the lipid interactions with key amino acids in proteins were responsible for major structure alterations, not just by modification of the catalytic site [4], but also by reaction with surface amino acids [2], which ultimately leads to structural (conformational) changes. Tandem mass spectrometry (MS/MS) studies performed on the peptidelipid Schiff and Michael adducts [2] report only product ions formed by peptide cleavages. Considering the formation of peptide/protein-lipid adducts, peptidephospholipid covalent interactions analogous to the ones described with 4-HNE could...

Heterogeneity of peptide adducts with carbonylated lipid peroxidation products

“…Reis et al . investigated in vitro the reactivity of 1‐palmitoyl‐2‐(9‐oxo‐nonanoyl)‐GPC with leucine enkephalin and found that the terminal amino group can form a Schiff base (+631.5 u) with PC‐bound aldehyde . In our previous study, we reported lysine to form Schiff base with 1‐palmitoyl‐2‐(9‐hydroxy‐12‐oxo‐dodecenoyl)‐GPC (+687.5 u) and Michael adducts with 1‐palmitoyl‐2‐(9‐keto‐12‐oxo‐dodecedienoyl)‐GPC (+703.5 u) and 1‐palmitoyl‐2‐(9‐keto‐10,12‐octadecadienoyl)‐GPC (+771.5 u) .…”

“…While alkanals can modify only primary amines through Schiff base formation, alkenals, alkadienals and hydroxy‐alkenals can additionally form Michael adducts with histidine imidazole and cysteine thiol . Protein modifications resulting from low molecular weight carbonyls were intensively studied during the last decade, while peptide and protein modifications by lipid‐bound carbonyls were only sporadically reported …”

“…[5,6] Protein modifications resulting from low molecular weight carbonyls were intensively studied during the last decade, [7][8][9][10][11][12] while peptide and protein modifications by lipid-bound carbonyls were only sporadically reported. [13][14][15] During the last decade, γ-hydroxyalkenals, in particular 4-hydroxy-2-alkenals, were recognized as one of the major sources of protein oxidative post translational modifications found in aging cells and oxidative stress-related pathological conditions. [16,17] By far the most commonly studied lipid peroxidation product is 4-hydroxy-2-nonenal (HNE), a product of n-6 PUFA oxidation, which was discovered in 1964 by Schauenstein et al [18] Successful synthesis of HNE in 1967 'opened the door' for many bioanalytical applications.…”

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

“…Carbonylated phospholipids (PL‐oxoLPP), that is, oxidized polyunsaturated fatty acids‐PL esters, are the dominant species of modified PL in vivo capable to modify nucleophilic substrates. In contrast to aliphatic oxoLPP, however, protein modifications resulting from lipid‐bound carbonyls have been rarely reported . The detection of protein‐oxoLPP in complex biological matrices relies on either specific immunoassays or sensitive mass spectrometry techniques, though the analysis of PL‐oxoLPP–protein adducts is challenged in reversed phase chromatography‐MS by the physicochemical properties, relatively low quantities in biological samples, and multiple positional isomers.…”

Radical peroxidation of palmitoyl-lineloyl-glycerophosphocholine liposomes: Identification of long-chain oxidised products by liquid chromatography–tandem mass spectrometry

“…On the other hand, phospholipids, present in cell membranes, low-density lipoproteins (LDL), cholesterol and triglycerides [2], playing an important role in membrane structure, metabolism and signal transduction [3], are also major targets of free radicals and have not been so extensively studied. Nonetheless, from the works published it has been inferred that the structural changes caused by radical peroxidation in phospholipids, such as phosphatidylcholines, affect the physical properties of the membrane bilayers [4,5] which are reflected in membrane integrity [6]; and are also responsible for covalent cross-linking between oxidised lipid moieties and proteins [7,8] leading to inactivation of membrane proteins and/or receptors [9]. In some cases, oxidised phospholipids show biological activity similar to platelet aggregating factors (PAF) [10], or may act as promoters in the expression of several genes, as proposed in the tumorogenesis of breast cancer cells [11].…”