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Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic β1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone., (Copyright © 2015 John Wiley & Sons, Ltd.)

Double bonds in polyunsaturated fatty acids (PUFA) and lipids are one of the major targets of reactive oxygen species (ROS). The resulting lipid peroxidation products (LPP) represent a group of chemically diverse compounds formed by several consecutive oxidative reactions. Oxidative cleavage leads to the formation of small aliphatic and lipid-bound aldehydes and ketones (oxoLPPs). These strong electrophiles can readily react with nucleophilic substrates, for example, side chains in proteins which can alter structure, function, and cellular distribution of the modified proteins. Despite growing interest in the field of oxidative lipidomics, only a few dominantly formed oxoLPP were identified. Due to the chemical and physical properties, aliphatic oxoLPPs are usually analyzed using gas chromatography-mass spectrometry (GC- MS), while nonvolatile lipid-bound oxoLPPs require liquid chromatography-mass spectrometry (LC-MS). To overcome the need for the two analyses, we have developed a new derivatization strategy to capture all oxoLPP independent to their properties with electrospray ionization (ESI) MS allowing simultaneous detection of aliphatic and lipid-bound oxoLPPs. Thus, the 7-(diethylamino)coumarin-3-carbohydrazide (CHH) derivatization reagent allowed us to identify 122 carbonyl compounds in a mixture of four PUFA and phosphatidylcholines (PC) oxidized in vitro.

Significance: Inflammation increases during the aging process. It is linked to mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Mitochondrial macromolecules are critical targets of oxidative damage; they contribute to respiratory uncoupling with increased ROS production, redox stress, and a cycle of senescence, cytokine production, and impaired oxidative phosphorylation. Targeting the formation or accumulation of oxidized biomolecules, particularly oxidized lipids, in immune cells and mitochondria could be beneficial for age-related inflammation and comorbidities. Recent Advances: Inflammation is central to age-related decline in health and exhibits a complex relationship with mitochondrial redox state and metabolic function. Improvements in mass spectrometric methods have led to the identification of families of oxidized phospholipids (OxPLs), cholesterols, and fatty acids that increase during inflammation and which modulate nuclear factor erythroid 2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor gamma (PPARγ), activator protein 1 (AP1), and NF-κB redox-sensitive transcription factor activity. Critical Issues: The kinetic and spatial resolution of the modified lipidome has profound and sometimes opposing effects on inflammation, promoting initiation at high concentration and resolution at low concentration of OxPLs. Future Directions: There is an emerging opportunity to prevent or delay age-related inflammation and vascular comorbidity through a resolving (oxy)lipidome that is dependent on improving mitochondrial quality control and restoring redox homeostasis. [ABSTRACT FROM AUTHOR]

Apoptosis is a key event in the control of inflammation. However, for this to be successful, dying cells must efficiently and effectively communicate their presence to phagocytes to ensure timely removal of dying cells. Here, we consider apoptotic cell-derived extracellular vesicles and the role of contained lipids and lipid mediators in ensuring effective control of inflammation. We discuss key outstanding issues in the study of cell death and cell communication, and introduce the concept of the 'active extracellular vesicle' as a metabolically active and potentially changing intercellular communicator. [ABSTRACT FROM AUTHOR]

Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris , it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology. Unlabelled Image • CD81 expressed in Pichia pastoris can be solubilised and purified using SMA polymer. • SMALP-encapsulated CD81 retains native folded structure. • Expression and buffer conditions can be optimized to improve protein quality. • The lipid environment surrounding CD81 is enriched with negatively charged lipids. [ABSTRACT FROM AUTHOR]