RESUMEN
Apoptosis is an essential process for normal physiology and plays a key role in the resolution of inflammation. Clearance of apoptotic cells (ACs) involves complex signalling between phagocytic cells, ACs, and the extracellular vesicles (EVs) they produce. Here, we discuss apoptotic cell-derived extracellular vesicles (ACdEVs) and how their structure relates to their function in AC clearance and the control of inflammation, focussing on the ACdEV proteome. We review the current knowledge, ongoing work and future directions for research in this field.
Asunto(s)
Apoptosis/fisiología , Vesículas Extracelulares/metabolismo , Animales , Apoptosis/genética , Humanos , Inflamación/metabolismo , Macrófagos/metabolismo , Fagocitosis/fisiología , Proteoma/metabolismo , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Comunicación Celular , Vesículas Extracelulares/metabolismo , Leucotrienos/metabolismo , Metabolismo de los Lípidos , Prostaglandinas/metabolismo , Apoptosis , Humanos , Inflamación/metabolismoRESUMEN
In recent years, there has been an increasing interest in nitro fatty acids (NO2-FA) as signaling molecules formed under nitroxidative stress. NO2-FA were detected in vivo in a free form, although it is assumed that they may also be esterified to phospholipids (PL). Nevertheless, insufficient discussion about the nature, origin, or role of nitro phospholipids (NO2-PL) was reported up to now. The aim of this study was to develop a mass spectrometry (MS) based approach which allows identifying nitroalkenes derivatives of three major PL classes found in living systems: phosphatidylcholines (PCs), phosphatidylethanolamine (PEs), and phosphatidylserines (PSs). NO2-PLs were generated by NO2BF4 in hydrophobic environment, mimicking biological systems. The NO2-PLs were then detected by electrospray ionization (ESI-MS) and ESI-MS coupled to hydrophilic interaction liquid chromatography (HILIC). Identified NO2-PLs were further analyzed by tandem MS in positive (as [M + H](+) ions for all PL classes) and negative-ion mode (as [M - H](-) ions for PEs and PSs and [M + OAc](-) ions for PCs). Typical MS/MS fragmentation pattern of all NO2-PL included a neutral loss of HNO2, product ions arising from the combined loss of polar headgroup and HNO2, [NO2-FA + H](+) and [NO2-FA - H](-) product ions, and cleavages on the fatty acid backbone near the nitro group, allowing its localization within the FA akyl chain. Developed MS method was used to identify NO2-PL in cardiac mitochondria from a well-characterized animal model of type 1 diabetes mellitus. We identified nine NO2-PCs and one NO2-PE species. The physiological relevance of these findings is still unknown.
Asunto(s)
Nitrocompuestos/análisis , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Fosfatidilserinas/análisis , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Masculino , Mitocondrias Cardíacas/química , Ratas Wistar , Espectrometría de Masas en TándemRESUMEN
Oxidized lipids play a significant role in the pathogenesis of numerous oxidative stress-related human disorders, such as atherosclerosis, obesity, inflammation, and autoimmune diseases. Lipid peroxidation, induced by reactive oxygen and nitrogen species, yields a high variety of modified lipids. Among them, carbonylated lipid peroxidation products (oxoLPP), formed by oxidation of the fatty acid moiety yielding aldehydes or ketones (carbonyl groups), are electrophilic compounds that are able to modify nucleophilic substrates like proteins, nucleic acid, and aminophospholipids. Some carbonylated phosphatidylcholines possess even pro-inflammatory activities. However, little is known about oxoLPP derived from other phospholipid (PL) classes. Here, we present a new analytical strategy based on the mass spectrometry (MS) of PL-oxoLPP derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). Shotgun MS revealed many oxoLPP derived from in vitro oxidized glycerophosphatidylglycerols (PG, 31), glycerophosphatidylcholine (PC, 23), glycerophosphatidylethanolamine (PE, 34), glycerophosphatidylserines (PS, 7), glycerophosphatidic acids (PA, 17), and phosphatidylinositiolphosphates (PIP, 6) vesicles. This data were used to optimize LipidXplorer-assisted identification, and a python-based post-processing script was developed to increase both throughput and accuracy. When applied to full lipid extracts from rat primary cardiomyocytes treated with peroxynitrite donor SIN-1, ten PL-bound oxoLPP were unambiguously identified by LC-MS, including two PC-, two PE-, one PG-, two PS-, and three PA-derived species. Some of the well-known carbonylated PC were detected, while most PL-oxoLPP were shown for the first time.
Asunto(s)
Peroxidación de Lípido , Fosfolípidos/química , Animales , Células Cultivadas , Cromatografía Liquida/métodos , Cumarinas/química , Humanos , Hidrazinas/química , Metabolómica/métodos , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Fosfolípidos/metabolismo , Ratas , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
The levels of nitro fatty acids (NO2-FA), such as nitroarachidonic, nitrolinoleic, nitrooleic, and dinitrooleic acids, are elevated under various inflammatory conditions, and this results in different anti-inflammatory effects. However, other multiply nitrated and nitro-oxidized FAs have not been studied so far. Owing to the low concentrations in vivo, NO2-FA analytics usually relies on targeted gas chromatography-tandem mass spectrometry (MS/MS) or liquid chromatography-MS/MS, and thus require standard compounds for method development. To overcome this limitation and increase the number and diversity of analytes, we performed in-depth mass spectrometry (MS) profiling of nitration products formed in vitro by incubating fatty acids with NO2BF4, and ONOO(-). The modified fatty acids were used to develop a highly specific and sensitive multiple reaction monitoring LC-MS method for relative quantification of 42 different nitrated and oxidized species representing three different groups: singly nitrated, multiply nitrated, and nitro-oxidized fatty acids. The method was validated in in vitro nitration kinetic studies and in a cellular model of nitrosative stress. NO2-FA were quantified in lipid extracts from 3-morpholinosydnonimine-treated rat primary cardiomyocytes after 15, 30, and 70 min from stress onset. The relatively high levels of dinitrooleic, nitroarachidonic, hydroxynitrodocosapenataenoic, nitrodocosahexaenoic, hydroxynitrodocosahexaenoic, and dinitrodocosahexaenoic acids confirm the presence of multiply nitrated and nitro-oxidized fatty acids in biological systems for the first time. Thus, in vitro nitration was successfully used to establish a targeted LC-MS/MS method that was applied to complex biological samples for quantifying diverse NO2-FA. Graphical Abstract Schematic representation of study design which combined in vitro nitration of different fatty acids, MS/MS characterization and optimization of MRM method for relative quantification, which was applied to follow dynamic of fatty acid nitration in cellular model of SIN-1 treated cardiomyoctes.
Asunto(s)
Ácidos Grasos/química , Nitratos/química , Animales , Células Cultivadas , Cromatografía Liquida , Cromatografía de Gases y Espectrometría de Masas , Miocitos Cardíacos/metabolismo , Nitrosación , Oxidación-Reducción , Ratas , Espectrometría de Masas en TándemRESUMEN
Reactive oxygen species (ROS) and other oxidative agents such as free radicals can oxidize polyunsaturated fatty acids (PUFA) as well as PUFA in lipids. The oxidation products can undergo consecutive reactions including oxidative cleavages to yield a chemically diverse group of products, such as lipid peroxidation products (LPP). Among them are aldehydes and ketones ("reactive carbonyls") that are strong electrophiles and thus can readily react with nucleophilic side chains of proteins, which can alter the protein structure, function, cellular distribution, and antigenicity. Here, we report a novel technique to specifically derivatize both low molecular and high molecular weight carbonylated LPP with 7-(diethylamino)coumarin-3-carbohydrazide (CHH) and analyze all compounds by electrospray ionization-mass spectrometry (ESI-MS) in positive ion mode. CHH-derivatized compounds were identified by specific neutral losses or fragment ions. The fragment ion spectra displayed additional signals that allowed unambiguous identification of the lipid, fatty acids, cleavage sites, and oxidative modifications. Oxidation of docosahexaenoic (DHA, 22:6), arachidonic (AA, 20:4), linoleic (LA, 18:2), and oleic acids (OA, 18:1) yielded 69 aliphatic carbonyls, whose structures were all deduced from the tandem mass spectra. When four phosphatidylcholine (PC) vesicles containing the aforementioned unsaturated fatty acids were oxidized, we were able to deduce the structures of 122 carbonylated compounds from the tandem mass spectra of a single shotgun analysis acquired within 15 min. The high sensitivity (LOD â¼ 1 nmol/L for 4-hydroxy-2-nonenal, HNE) and a linear range of more than 3 orders of magnitude (10 nmol/L to 10 µmol/L for HNE) will allow further studies on complex biological samples including plasma.
Asunto(s)
Aldehídos/análisis , Cetonas/análisis , Peroxidación de Lípido , Espectrometría de Masa por Ionización de Electrospray , Ácidos Araquidónicos/química , Cumarinas/química , Ácidos Docosahexaenoicos/química , Hidrazinas/química , Ácidos Linoleicos/química , Peso Molecular , Ácidos Oléicos/química , Oxidación-Reducción , Fosfatidilcolinas/química , Especies Reactivas de Oxígeno/químicaRESUMEN
The c-Jun N-terminal kinases (JNKs) are a family of proteins that, once activated by stress stimuli, can alter neuronal functions and survival. The JNK cascade plays a crucial role in the post-synaptic neuronal compartment by altering its structural organization and leading, at worst, to an overall impairment of neuronal communication. Increasing evidence suggests that synaptic impairment is the first neurodegenerative event in Alzheimer's disease (AD). To better elucidate this mechanism, we longitudinally studied 5xFAD mice at three selected time points representative of human AD symptom progression. We tested the mice cognitive performance by using the radial arm water maze (RAWM) in parallel with biochemical evaluations of post-synaptic enriched protein fraction and total cortical parenchyma. We found that 5xFAD mice presented a strong JNK activation at 3.5 months of age in the post-synaptic enriched protein fraction. This JNK activation correlates with a structural alteration of the post-synaptic density area and with memory impairment at this early stage of the disease that progressively declines to cause cell death. These findings pave the way for future studies on JNK as a key player in early neurodegeneration and as an important therapeutic target for the development of new compounds able to tackle synaptic impairment in the early phase of AD pathology.
Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Proteínas Quinasas JNK Activadas por Mitógenos , Animales , Ratones , Enfermedad de Alzheimer/metabolismo , Disfunción Cognitiva/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Fosforilación , Modelos Animales de EnfermedadRESUMEN
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.
Asunto(s)
Inflamación/metabolismo , Metabolismo de los Lípidos , Peroxidación de Lípido , Oxidación-Reducción , Animales , Biomarcadores , Citocinas/metabolismo , Susceptibilidad a Enfermedades/inmunología , Humanos , Inflamación/etiología , Inflamación/patología , Mitocondrias/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Fascioliasis is a neglected zoonotic disease that infects humans and ruminant species worldwide. In the absence of vaccines, control of fascioliasis is primarily via anthelminthic treatment with triclabendazole (TCBZ). Parasitic flatworms, including Fasciola hepatica, are active secretors of extracellular vesicles (EVs), but research has not been undertaken investigating EV anthelmintic sequestration. Adult F. hepatica were cultured in lethal and sub-lethal doses of TCBZ and its active metabolites, in order to collect EVs and evaluate their morphological characteristics, production and anthelmintic metabolite content. Transmission electron microscopy demonstrated that F. hepatica exposed to TCBZ and its metabolites produced EVs of similar morphology, compared to non-TCBZ exposed controls, even though TCBZ dose and/or TCBZ metabolite led to measurable structural changes in the treated F. hepatica tegument. qNano particle analysis revealed that F. hepatica exposed to TCBZ and its metabolites produced at least five times greater EV concentrations than non-TCBZ controls. A combined mass spectrometry and qNano particle analysis confirmed the presence of TCBZ and the TCBZ-sulphoxide metabolite in anthelmintic exposed EVs, but limited TCBZ sulphone was detectable. This data suggests that EVs released from adult F. hepatica have a biological role in the sequestration of TCBZ and additional toxic xenobiotic metabolites.
Asunto(s)
Fasciola hepatica/metabolismo , Triclabendazol/metabolismo , Triclabendazol/farmacología , Animales , Antihelmínticos/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Fascioliasis/tratamiento farmacológico , Ovinos , Enfermedades de las Ovejas/parasitología , Triclabendazol/uso terapéutico , Zoonosis/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Modelos Moleculares , Pliegue de Proteína , Tetraspanina 28/química , Cristalografía por Rayos X , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales , Tetraspanina 28/genética , Tetraspanina 28/metabolismoRESUMEN
The airways of individuals with cystic fibrosis (CF) are abundantly colonised by Staphylococcus aureus and Pseudomonas aeruginosa. Co-infecting hypoxic regions of static mucus within CF airways, together with decreases in pulmonary function, mucus plugging and oxygen consumption by host neutrophils gives rise to regions of anoxia. This study determined the impact of anaerobiosis upon S. aureus-P. aeruginosa interactions in planktonic co-culture and mixed species biofilms in vitro. Whilst anoxia reduced the ability for P. aeruginosa CF isolates to dominate over S. aureus, this occurred in an isolate dependent manner. Investigations into the underlying mechanisms suggest that the anti-staphylococcal compound facilitating P. aeruginosa dominance under normoxia and anoxia is greater than 3 kDa in size and is heat-stable. Not all interspecies interactions studied were antagonistic, as S. aureus exoproducts were shown to restore and enhance P. aeruginosa motility under normoxia and anoxia in an isolate dependent manner. Collectively, this study suggests changes in oxygen availability within regions of the CF lung is likely to influence interspecies interactions and in turn, potentially influence disease progression.
Asunto(s)
Anaerobiosis , Fibrosis Quística/complicaciones , Interacciones Microbianas , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/fisiología , Biopelículas , Técnicas de Cocultivo , Humanos , Hipoxia , Consumo de Oxígeno , Plancton , Pseudomonas aeruginosa/patogenicidad , VirulenciaRESUMEN
Oxysterols (OHC) are biologically active cholesterol metabolites circulating in plasma that may be formed enzymatically (e.g. 24S-OHC, 25-OHC and 27-OHC) or by autoxidative mechanisms (e.g. 7-ketocholesterol, 7ß-OHC and 25-OHC). Oxysterols are more soluble than cholesterol and are reported to exert inflammatory, cytoprotective and apoptotic effects according to concentration and species. Esterified oxysterols have been analysed in people with dementia and cardiovascular diseases although there is no consistent relationship between oxysterol esters and disease. However, oxysterol esters are held in lipoprotein core and may not relate to the concentration and activity of plasma free oxysterols. Methodological limitations have challenged the analysis of free oxysterols to date. We have developed a fast, sensitive and specific quantitative LC-MS/MS, multiple reaction monitoring (MRM) method to target five oxysterols in human plasma with analyte recoveries between 72% and 82% and sensitivities between 5 and 135â¯pg/ml. A novel method was used to investigate the hypothesis that simvastatin may reduce the concentrations of specific plasma free oxysterols in hypercholesterolaemia. Twenty healthy male volunteers were recruited (aged 41-63 years); ten were asymptomatic with high plasma cholesterol >â¯6.5â¯mM and ten were healthy with normal plasma cholesterol (<â¯6.5â¯mM). Simvastatin (40â¯mg/day) was prescribed to those with hypercholesterolaemia. Plasma samples were taken from both groups at baseline and after three months. Simvastatin reduced plasma cholesterol by ~35% (pâ¯<â¯0.05) at the end of three months. Oxysterols generated by autoxidation (but not enzymatically) were elevated up to 45 fold in hypercholesterolaemic midlife men. Plasma oxysterols were restored to those of healthy controls after simvastatin intervention suggesting that autoxidation is either prevented by simvastatin directly or that autoxidation is less prevalent when plasma cholesterol concentrations are within the normal range.
Asunto(s)
Colesterol/metabolismo , Hipercolesterolemia/tratamiento farmacológico , Oxiesteroles/sangre , Simvastatina/administración & dosificación , Adulto , Cromatografía Liquida , Humanos , Hipercolesterolemia/sangre , Hipercolesterolemia/patología , Masculino , Persona de Mediana Edad , Oxiesteroles/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.
Asunto(s)
Vesículas Extracelulares/química , Carbohidratos , Humanos , Lípidos , Ácidos Nucleicos , Pronóstico , ProteínasRESUMEN
Periodontal disease is a prevalent chronic inflammatory condition characterised by an aberrant host response to a pathogenic plaque biofilm resulting in local tissue damage and frustrated healing that can result in tooth loss. Cysteine proteases (gingipains) from the key periodontal pathogen Porphyromonas gingivalis have been implicated in periodontal disease pathogenesis by inhibiting inflammation resolution and are linked with systemic chronic inflammatory conditions such as rheumatoid arthritis. Efficient clearance of apoptotic cells is essential for the resolution of inflammation and tissue restoration. Here we sought to characterise the innate immune clearance of apoptotic cells and its modulation by gingipains. We examined the capacity of gingipain-treated macrophages to migrate towards and phagocytose apoptotic cells. Lysine gingipain treatment of macrophages impaired macrophage migration towards apoptotic neutrophils. Furthermore, lysine gingipain treatment reduced surface expression levels of CD14, a key macrophage receptor for apoptotic cells, which resulted in reduced macrophage interactions with apoptotic cells. Additionally, while apoptotic cells and their derived secretome were shown to inhibit TNF-α-induced expression by P. gingivalis lipopolysaccharide, we demonstrated that gingipain preparations induced a rapid inflammatory response in macrophages that was resistant to the anti-inflammatory effects of apoptotic cells or their secretome. Taken together, these data indicate that P. gingivalis may promote the chronic inflammation seen in periodontal disease patients by multiple mechanisms, including rapid, potent gingipain-mediated inflammation, coupled with receptor cleavage leading to defective clearance of apoptotic cells and reduced anti-inflammatory responses. Thus, gingipains represent a potential therapeutic target for intervention in the management of chronic periodontal disease.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Apoptosis/fisiología , Movimiento Celular/fisiología , Cisteína Endopeptidasas/metabolismo , Macrófagos/patología , Neutrófilos/patología , Fagocitosis/fisiología , Porphyromonas gingivalis/metabolismo , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Biopelículas/crecimiento & desarrollo , Línea Celular Tumoral , Proteasas de Cisteína/metabolismo , Cisteína-Endopeptidasas Gingipaínas , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Peroxidación de Lípido , Lípidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Aldehídos/química , Cumarinas/química , Hidrazinas/química , Fosfolípidos/química , Estadística como AsuntoRESUMEN
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 ß1 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.
Asunto(s)
Lípidos/química , Espectrometría de Masas/métodos , Modelos Químicos , Péptidos/química , Apolipoproteína B-100/química , Isomerismo , Fosfatidilcolinas/químicaRESUMEN
Highly reactive lipid peroxidation-derived carbonyls (oxoLPP) modify protein nucleophiles via Michael addition or Schiff base formation. Once formed, Michael adducts can be further stabilized via cyclic hemiacetals with or without loss of water. Depending on the mechanism of their formation, peptide-oxoLPP can carry aldehyde or keto groups and thus be a part of the total protein carbonylation level. If a carbonyl function is lost during consecutive reactions, the oxoLPP-peptide adducts will not be detected using the common carbonyl labeling protocols. Because of the differences in adduct stabilities, it is possible to address the heterogeneity of peptide/protein-oxoLPP adducts by careful evaluation of tandem mass spectra of modified peptides. Here, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of lysine, cysteine and histidine containing model peptides co-incubated with oxidized 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine to characterize the collision-induced dissociation behavior of peptide-carbonyl adducts. Numerous modifications were detected based on the analysis of tandem mass spectra, including Schiff bases on lysine (two), Michael adducts on lysine (six), cysteine (eleven) and histidine (two), as well as 4-hydroxy-2-aldehydes derived dehydrated cyclic hemiacetals on cysteine (five) and histidine (one). Additionally, cysteine and histidine side chains were modified by lipid-bound aldehydes as Michael adducts and dehydrated hemiacetals. The tandem mass spectra revealed collision-induced dissociation characteristics specific for each class of oxoLPP-peptide adducts.
Asunto(s)
Peroxidación de Lípido , Péptidos/análisis , Péptidos/química , Carbonilación Proteica , Aldehídos/química , Cromatografía Liquida , Espectrometría de Masas en TándemRESUMEN
This report focuses on studies of lipid peroxidation products reactivity towards the side chains of cysteine, histidine, and lysine residues in structurally unordered peptides. Thus we have analyzed linoleic acid peroxidation products (LaPP) obtained by incubating 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine (PLPC) overnight with or without H(2)O(2) in the presence or absence of CuCl. In total, 55 different LaPP were identified with 26 containing reactive carbonyl groups. The strongest oxidation conditions (H(2)O(2) and Cu(I), i.e. a Fenton-like reagent) yielded 51 LaPP, whereas air oxidation produced only 12 LaPP. Independent of the oxidation conditions, around half of all LaPP were short-chain (oxidative cleavage) and the others long-chain (oxygen addition) PLPC oxidation products. The stronger oxidation conditions increased the number of LaPP, but also oxidized the added peptide Ac-PAAPAAPAPAEXTPV-OH (X=Cys, His or Lys) very quickly, especially under Fenton conditions. Thus, PLPC was oxidized by milder conditions (air or Cu(I)), incubated with the peptide and the peptide modifications were then analyzed by nano-RPC-ESI-Orbitrap-MS. Ten LaPP-derived peptide modifications were identified at lysine, whereas nine products were identified for cysteine and only three for histidine. Three high molecular weight LaPP still esterified to the GPC backbone were detected on Lys-containing peptide. Furthermore, three LaPP-derived mass shifts were obtained at cysteine, which have not previously been reported.