Peptide-Phospholipid Cross-Linking Reactions: Identification of Leucine Enkephalin-Alka(e)nal-Glycerophosphatidylcholine Adducts by Tandem Mass Spectrometry

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 peptide-glycerophosphatidylcholine adducts

Halophyte plants from sustainable marine aquaponics are a valuable source of omega-3 polar lipids

Marine aquaponics is a promising sustainable approach for the production of profitable crops such as halophytes. However, the effect of this culture approach on the lipid composition of halophytes remains unknown. In this work, we contrasted the polar lipidome of Salicornia ramosissima and Halimione portulacoides when produced in marine aquaponics (effluent from a super-intensive flatfish aquaculture production), with that of conspecifics from donor wild populations. Phospholipids and glycolipids were identified and quantified by LC-MS and MS/MS and their profile statistically analysed. Halophytes produced in aquaponics have higher levels of glycolipids with n-3 fatty acids (DGDG 36:3; SQDG 36:3; MGDG 36:6) compared with the donor wild populations. In the case of H. portulacoides, a significant increase of phospholipids bearing n-3 fatty acids (most in PC and PE) was also recorded. These lipids have potential applications in food, feed and pharmaceutical industries, contributing to the valorization of halophytes produced under sustainable aquaculture practices.publishe

Discovery of bioactive nitrated lipids and nitro-lipid-protein adducts using mass spectrometry-based approaches

Nitro-fatty acids (NO2-FA) undergo reversible Michael adduction reactions with cysteine and histidine residues leading to the post-translational modification (PTM) of proteins. This electrophilic character of NO2-FA is strictly related to their biological roles. The NO2-FA-induced PTM of signaling proteins can lead to modifications in protein structure, function, and subcellular localization. The nitro lipid-protein adducts trigger a series of downstream signaling events that culminates with anti-inflammatory, anti-hypertensive, and cytoprotective effects mediated by NO2-FA. These lipoxidation adducts have been detected and characterized both in model systems and in biological samples by using mass spectrometry (MS)-based approaches. These MS approaches allow to unequivocally identify the adduct together with the targeted residue of modification. The identification of the modified proteins allows inferring on the possible impact of the NO2-FA-induced modification. This review will focus on MS-based approaches as valuable tools to identify NO2-FA-protein adducts and to unveil the biological effect of this lipoxidation adducts.publishe

Liquid chromatography/tandem mass spectrometry characterization of nitroso, nitrated and nitroxidized cardiolipin products

Cardiolipins (CL) are anionic dimeric phospholipids bearing four fatty acids, found in inner mitochondrial membrane as structural components and are involved in several processes as oxidative phosphorylation or apoptotic signalling. As other phospholipids, CL can be modified by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can modulate various cellular functions. Modifications of CL by RNS remain largely unstudied although other nitrated lipids are emerging as bioactive molecules. In this work, we developed a C30-LC-HRMS/MS methodology to identify the nitrated and nitroxidized tetralinoleoyl-cardiolipin (TLCL), using a biomimetic model of nitration, and to disclose specific fragmentation pathways under HCD MS/MS. Using this lipidomics approach, we were able to separate and identify nitro, nitroso, nitronitroso, and nitroxidized TLCL derivatives, comprising 11 different nitrated compounds. These products were identified using accurate mass measurements and the fragmentation pattern acquired in higher-energy collision dissociation (HCD)-tandem MS/MS experiments. These spectra showed classifying fragmentation pathways, yielding phosphatidic acid (PA-), lysophosphatidic acid (LPA-), and carboxylate fragment ions with the modifying moiety. Remarkably, the typical neutral losses associated with the added moieties were not observed. In conclusion, this work has developed a new method for the identification of nitroso, nitrated and nitroxidized cardiolipin products by using a C30LC-MS platform method, potentially allowing their detection in biological samples.publishe

Analysis of oxidised and glycated aminophospholipids: complete structural characterisation by C30 liquid chromatography-high resolution tandem mass spectrometry

The aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS) are widely present in cell membranes and lipoproteins. Glucose and reactive oxygen species (ROS), such as the hydroxyl radical (•OH), can react with APL leading to an array of oxidised, glycated and glycoxidised derivatives. Modified APL have been implicated in inflammatory diseases and diabetes, and were identified as signalling molecules regulating cell death. However, the biological relevance of these molecules has not been completely established, since they are present in very low amounts, and new sensitive methodologies are needed to detect them in biological systems. Few studies have focused on the characterisation of APL modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS), mainly using C5 or C18 reversed phase (RP) columns. In the present study, we propose a new analytical approach for the characterisation of complex mixtures of oxidised, glycated and glycoxidised PE and PS. This LC approach was based on a reversed-phase C30 column combined with high-resolution MS, and higher energy C-trap dissociation (HCD) MS/MS. C30 RP-LC separated short and long fatty acyl oxidation products, along with glycoxidised APL bearing oxidative modifications on the glucose moiety and the fatty acyl chains. Functional isomers (e.g. hydroxy-hydroperoxy-APL and tri-hydroxy-APL) and positional isomers (e.g. 9-hydroxy-APL and 13-hydroxy-APL) were also discriminated by the method. HCD fragmentation patterns allowed unequivocal structural characterisation of the modified APL, and are translatable into targeted MS/MS fingerprinting of the modified derivatives in biological samples.publishe

Serum phospholipidomics reveals altered lipid profile and promising biomarkers in multiple sclerosis

Multiple sclerosis is a neurodegenerative disease causing disability in young adults. Alterations in metabolism and lipid profile have been associated with this disease. Several studies have reported changes in the metabolism of arachidonic acid and the profile of fatty acids, ceramides, phospholipids and lipid peroxidation products. Nevertheless, the understanding of the modulation of circulating lipids at the molecular level in multiple sclerosis remains unclear. In the present study, we sought to assess the existence of a distinctive lipid signature of multiple sclerosis using an untargeted lipidomics approach. It also aimed to assess the differences in lipid profile between disease status (relapse and remission). For this, we used hydrophilic interaction liquid chromatography coupled with mass spectrometry for phospholipidomic profiling of serum samples from patients with multiple sclerosis. Our results demonstrated that multiple sclerosis has a phospholipidomic signature different from that of healthy controls, especially the PE, PC, LPE, ether-linked PE and ether-linked PC species. Plasmalogen PC and PE species, which are natural endogenous antioxidants, as well as PC and PE polyunsaturated fatty acid esterified species showed significantly lower levels in patients with multiple sclerosis and patients in both remission and relapse of multiple sclerosis. Our results show for the first time that the serum phospholipidome of multiple sclerosis is significantly different from that of healthy controls and that few phospholipids, with the lowest p-value, such as PC(34:3), PC(36:6), PE(40:10) and PC(38:1) may be suitable as biomarkers for clinical applications in multiple sclerosis.publishe

Modulation of the inflammatory response of immune cells in human peripheral blood by oxidized arachidonoyl aminophospholipids

Aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS), can be oxidized upon oxidative stress. Oxidized PE and PS have been detected in clinical samples of different pathologies and may act as modulators of the inflammatory response. However, few studies have focused on the effects of oxidized APL (ox-APL) esterified with arachidonic acid, even though a considerable number of studies have assessed the modulation of the immune system by oxidized 1-palmitoyl-2-arachidonoyl-sn-3-glycerophosphocholine (OxPAPC). In the present study, we have used flow cytometry to evaluate the ability of oxidized PAPE (OxPAPE) and PAPS (OxPAPS) to promote or suppress an inflammatory phenotype on monocytes subsets and myeloid dendritic cells (mDCs). The results indicate that OxPAPE increases the frequency of all monocyte subpopulations expressing TNF-α, which promotes an inflammatory response. However, immune cell stimulation with OxPAPE in the presence of LPS results in a decrease of TNF-α expressed by classical monocytes. Incubation with OxPAPS and LPS induces a decrease in TNF-α produced by monocytes, and a significant decrease in IL-1β expressed by monocytes and mDCs, indicating that OxPAPS reduces the LPS-induced pro-inflammatory expression in these populations. These results show the importance of OxPAPE and OxPAPS as modulators of the inflammatory response and demonstrate their possible contribution to the onset and resolution of human diseases related to oxidative stress and inflammation.publishe

Current trends in the traceability of geographic origin and detection of species-mislabeling in marine bivalves

Marine bivalves are increasingly consumed worldwide, with their complex supply chain being particularly prone to fraud. This scenario drives economic losses and is a threat to public health, with multiple recent food worries driving consumers to demand more transparency and information on the seafood they buy. To increase consumers confidence in bivalves and enforce current legislation, robust tools are needed to fight species mislabeling and confirm the place of origin of bivalves being traded. The present study provides a critical overview based on a databases search, over the traceability of geographic origin and detection of species-mislabeling in marine bivalves, summarizing the tools currently available to confirm claims on these topics along the supply chain. We also identify current trends on the use of tools, pinpoint which countries contribute to advance the state of the art on these topics, and highlight the bivalve groups/species being more commonly surveyed. The most used tools to expose species mislabeling in marine bivalves are DNA and fatty acid analysis, while elemental analysis is the most commonly employed approach to confirm their geographic origin. Stable and unstable isotope analysis, as well as metabolomics, are also starting to be increasingly used to verify species authenticity and provenance in marine bivalves. Further studies are still needed to identify annual/seasonal variations and determine if these can be a constraint for the optimization of protocols to fight fraudulent practices. The implementation of an open global database to allow realtime data comparison will be paramount to advance the state of the art.publishe

Plasma phospholipidomic profile differs between children with phenylketonuria and healthy children

Phenylketonuria (PKU) is a disease of the catabolism of phenylalanine (Phe), caused by an impaired function of the enzyme phenylalanine hydroxylase. Therapeutics is based on the restriction of Phe intake, which mostly requires a modification of the diet. Dietary restrictions can lead to imbalances in specific nutrients, including lipids. In the present study, the plasma phospholipidome of PKU and healthy children (CT) was analysed by HILIC-MS/MS and GC-MS. Using this approach, 187 lipid species belonging to 9 different phospholipid classes and 3 ceramides were identified. Principal component analysis of the lipid species dataset showed a distinction between PKU and CT groups. Univariate analysis revealed that 146 species of phospholipids were significantly different between both groups. Lipid species showing significant variation included phosphatidylcholines, containing polyunsaturated fatty acids (PUFA), which were more abundant in PKU. The high level of PUFA-containing lipid species in children with PKU may be related to a diet supplemented with PUFA. This study was the first report comparing the plasma polar lipidome of PKU and healthy children, highlighting that the phospholipidome of PKU children is significantly altered compared to CT. However, further studies with larger cohorts are needed to clarify whether these changes are specific to phenylketonuric children.publishe

Lipidomic signature of Bacillus licheniformis I89 during the different growth phases unravelled by high-resolution liquid chromatography-mass spectrometry

Bacillus licheniformis I89 is a non-pathogenic, Gram-positive bacterium, frequently found in soil. It has several biotechnological applications as producer of valuable compounds such as proteases, amylases, surfactants, and lantibiotics. Herein, it is reported the identification of the polar lipidome of B. licheniformis I89 during the different growth phases (lag, exponential and stationary) at 37 °C. The analytical approach relied on hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry (HILIC-ESI-MS), accurate mass measurements and tandem mass spectrometry (MS/MS). In the lipidome of B. licheniformis I89 were identified four phospholipid classes: phosphatidylethanolamine, phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin; two glycolipid classes: monoglycosyldiacylglycerol and diglycosyldiacylglycerol; and two phosphoglyceroglycolipid classes: mono-alanylated lipoteichoic acid primer and lipoteichoic acid primer. The same lipid species were identified at the different growth phases, but there were significant differences on the relative abundance of some molecular species. There was a significant increase in the 30:0 lipid species and a significant decrease in the 32:0 lipid species, between exponential and stationary phases, when compared to lag phase. No differences were observed between exponential and stationary phases. The lipidomic-based approach used herein is a very promising tool to be employed in the study of bacterial lipid composition, which is a requirement to understand its metabolism and response to growth conditions.publishe