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1.
Nat Methods ; 20(2): 193-204, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36543939

RESUMO

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.


Assuntos
Biologia Computacional , Lipidômica , Biologia Computacional/métodos , Software , Informática , Lipídeos/química
2.
Metabolomics ; 17(6): 55, 2021 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-34091802

RESUMO

BACKGROUND: Improvements in mass spectrometry (MS) technologies coupled with bioinformatics developments have allowed considerable advancement in the measurement and interpretation of lipidomics data in recent years. Since research areas employing lipidomics are rapidly increasing, there is a great need for bioinformatic tools that capture and utilize the complexity of the data. Currently, the diversity and complexity within the lipidome is often concealed by summing over or averaging individual lipids up to (sub)class-based descriptors, losing valuable information about biological function and interactions with other distinct lipids molecules, proteins and/or metabolites. AIM OF REVIEW: To address this gap in knowledge, novel bioinformatics methods are needed to improve identification, quantification, integration and interpretation of lipidomics data. The purpose of this mini-review is to summarize exemplary methods to explore the complexity of the lipidome. KEY SCIENTIFIC CONCEPTS OF REVIEW: Here we describe six approaches that capture three core focus areas for lipidomics: (1) lipidome annotation including a resolvable database identifier, (2) interpretation via pathway- and enrichment-based methods, and (3) understanding complex interactions to emphasize specific steps in the analytical process and highlight challenges in analyses associated with the complexity of lipidome data.


Assuntos
Biologia Computacional , Lipidômica , Bases de Dados Factuais , Lipídeos , Espectrometria de Massas
5.
Anal Chem ; 89(17): 8800-8807, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28753264

RESUMO

Lipids are dynamic constituents of biological systems, rapidly responding to any changes in physiological conditions. Thus, there is a large interest in lipid-derived markers for diagnostic and prognostic applications, especially in translational and systems medicine research. As lipid identification remains a bottleneck of modern untargeted lipidomics, we developed LipidHunter, a new open source software for the high-throughput identification of phospholipids in data acquired by LC-MS and shotgun experiments. LipidHunter resembles a workflow of manual spectra annotation. Lipid identification is based on MS/MS data analysis in accordance with defined fragmentation rules for each phospholipid (PL) class. The software tool matches product and neutral loss signals obtained by collision-induced dissociation to a user-defined white list of fatty acid residues and PL class-specific fragments. The identified signals are tested against elemental composition and bulk identification provided via LIPID MAPS search. Furthermore, LipidHunter provides information-rich tabular and graphical reports allowing to trace back key identification steps and perform data quality control. Thereby, 202 discrete lipid species were identified in lipid extracts from rat primary cardiomyocytes treated with a peroxynitrite donor. Their relative quantification allowed the monitoring of dynamic reconfiguration of the cellular lipidome in response to mild nitroxidative stress. LipidHunter is available free for download at https://bitbucket.org/SysMedOs/lipidhunter .


Assuntos
Biologia Computacional/métodos , Fosfolipídeos/sangue , Software , Animais , Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Espectrometria de Massas/métodos , Miócitos Cardíacos/química , Ratos
6.
Anal Bioanal Chem ; 407(17): 5161-73, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25701423

RESUMO

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.


Assuntos
Peroxidação de Lipídeos , Fosfolipídeos/química , Animais , Células Cultivadas , Cromatografia Líquida/métodos , Cumarínicos/química , Humanos , Hidrazinas/química , Metabolômica/métodos , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Fosfolipídeos/metabolismo , Ratos , Espectrometria de Massas por Ionização por Electrospray/métodos
7.
Nat Commun ; 13(1): 6547, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36319635

RESUMO

Lipids are a structurally diverse class of biomolecules which can undergo a variety of chemical modifications. Among them, lipid (per)oxidation attracts most of the attention due to its significance in the regulation of inflammation, cell proliferation and death programs. Despite their apparent regulatory significance, the molecular repertoire of oxidized lipids remains largely elusive as accurate annotation of lipid modifications is complicated by their low abundance and often unknown, biological context-dependent structural diversity. Here, we provide a workflow based on the combination of bioinformatics and LC-MS/MS technologies to support identification and relative quantification of oxidized complex lipids in a modification type- and position-specific manner. The developed methodology is used to identify epilipidomics signatures of lean and obese individuals with and without type 2 diabetes. The characteristic signature of lipid modifications in lean individuals, dominated by the presence of modified octadecanoid acyl chains in phospho- and neutral lipids, is drastically shifted towards lipid peroxidation-driven accumulation of oxidized eicosanoids, suggesting significant alteration of endocrine signalling by oxidized lipids in metabolic disorders.


Assuntos
Diabetes Mellitus Tipo 2 , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Fluxo de Trabalho , Lipídeos/química , Plasma/química
8.
J Invest Dermatol ; 142(1): 4-11.e1, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34924150

RESUMO

Although lipids are crucial molecules for cell structure, metabolism, and signaling in most organs, they have additional specific functions in the skin. Lipids are required for the maintenance and regulation of the epidermal barrier, physical properties of the skin, and defense against microbes. Analysis of the lipidome-the totality of lipids-is of similar complexity to those of proteomics or other omics, with lipid structures ranging from simple, linear, to highly complex structures. In addition, the ordering and chemical modifications of lipids have consequences on their biological function, especially in the skin. Recent advances in analytic capability (usually with mass spectrometry), bioinformatic processing, and integration with other dermatological big data have allowed researchers to increasingly understand the roles of specific lipid species in skin biology. In this paper, we review the techniques used to analyze skin lipidomics and epilipidomics.


Assuntos
Lipidômica/métodos , Pele/metabolismo , Animais , Big Data , Pesquisa Biomédica , Biologia Computacional , Epigênese Genética , Humanos , Metabolismo dos Lipídeos , Espectrometria de Massas , Pele/patologia
9.
Cell Rep Med ; 2(10): 100407, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34755127

RESUMO

Obesity, characterized by expansion and metabolic dysregulation of white adipose tissue (WAT), has reached pandemic proportions and acts as a primer for a wide range of metabolic disorders. Remodeling of WAT lipidome in obesity and associated comorbidities can explain disease etiology and provide valuable diagnostic and prognostic markers. To support understanding of WAT lipidome remodeling at the molecular level, we provide in-depth lipidomics profiling of human subcutaneous and visceral WAT of lean and obese individuals. We generate a human WAT reference lipidome by performing tissue-tailored preanalytical and analytical workflows, which allow accurate identification and semi-absolute quantification of 1,636 and 737 lipid molecular species, respectively. Deep lipidomic profiling allows identification of main lipid (sub)classes undergoing depot-/phenotype-specific remodeling. Previously unanticipated diversity of WAT ceramides is now uncovered. AdipoAtlas reference lipidome serves as a data-rich resource for the development of WAT-specific high-throughput methods and as a scaffold for systems medicine data integration.


Assuntos
Tecido Adiposo Branco/metabolismo , Lipidômica , Idoso , Calibragem , Ceramidas/química , Ceramidas/metabolismo , Fracionamento Químico , Etanolaminas/química , Etanolaminas/metabolismo , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Lipídeos/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Fenótipo , Plasmalogênios/metabolismo , Triglicerídeos/metabolismo , Regulação para Cima
10.
F1000Res ; 10: 4, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33564392

RESUMO

Lipidomics increasingly describes the quantification using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues.


Assuntos
Lipidômica , Lipídeos , Animais , Internet , Metabolismo dos Lipídeos , Redes e Vias Metabólicas
11.
J Am Soc Mass Spectrom ; 32(7): 1798-1809, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34096708

RESUMO

Vegetables oils, rich in polyunsaturated fatty acids, are vulnerable to oxidation during manufacturing, processing, and food preparation. Currently, individual oxidation products are not well characterized, and hence, the health impacts of these unique lipid species remain unknown. Here, we introduce an extensive oxidized lipidomics in silico tandem mass spectrometry library and integrate these libraries within a user-friendly software covering a comprehensive redox lipidomics workflow. We apply this workflow to olive, soy, and walnut cooking oil; comparing unheated oil, oil after deep frying potatoes, and oil after oven frying potatoes. We annotated over a thousand oxidized triglycerides across 273 features (many coeluted). This software was validated against traditional chemical assays of oxidation, known oxidized lipids in castor oil, synthesized standards, and an alternate software LPPtiger. Development of these new software programs for redox lipidomics opens the door to characterize health implications of individual oxidation products.


Assuntos
Culinária , Lipidômica/métodos , Óleos de Plantas , Solanum tuberosum/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida , Oxirredução , Óleos de Plantas/análise , Óleos de Plantas/química
12.
Chem Phys Lipids ; 226: 104786, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31229410

RESUMO

Cold atmospheric plasma (CAP) is an emerging source for the locally defined delivery of reactive species, and its clinical potential has been identified in the control of inflammatory processes, such as acute and chronic wounds, or cancerous lesions. Lipids, due to their localization and chemical structure as ideal targets for oxidative species, are relevant modifiers of physiological processes. Human forehead lipids collected on a target were treated by an argon plasma jet and immediately analyzed by direct-infusion high-resolution tandem mass spectrometry (DI-MS2) or liquid chromatography-tandem MS (RP-LC/MS2). Subsequent data analysis was performed by LipidHunter (University of Leipzig), LipidXplorer (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden), and LipidSearch (Thermo Scientific). With either MS method, all major lipid classes of sebum lipids were detected. Significant differences regarding triacylglycerols (predominantly identified in RP-LC/MS2) and ceramides (predominantly identified in DI-MS2) indicate experimental- or approach-inherent distinctions. A CAP-driven oxidation of triacyclglycerols, ceramides, and cholesteryl esters was detected such as truncations and hydroperoxylations, but at a significantly lower extent than expected. Scavenging of reactive species due to naturally present antioxidants in the samples and the absence of a liquid interphase to allow reactive species deposition by the CAP will have contributed to the limited amount of oxidation products observed. In addition, limitations of the software's capability of identifying unexpected oxidized lipids potentially led to an underestimation of the CAP impact on skin lipids, indicating a need for further software development. With respect to the clinical application of CAP, the result indicates that intact skin with its sebum/epidermal lipid overlay is well protected and that moderate treatment will yield limited (if any) functional consequences in the dermal tissue.


Assuntos
Lipídeos/química , Gases em Plasma/química , Pele/química , Adulto , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Espectrometria de Massas em Tandem , Adulto Jovem
13.
Free Radic Biol Med ; 144: 110-123, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31035005

RESUMO

The high chemical diversity of lipids allows them to perform multiple biological functions ranging from serving as structural building blocks of biological membranes to regulation of metabolism and signal transduction. In addition to the native lipidome, lipid species derived from enzymatic and non-enzymatic modifications (the epilipidome) make the overall picture even more complex, as their functions are still largely unknown. Oxidized lipids represent the fraction of epilipidome which has attracted high scientific attention due to their apparent involvement in the onset and development of numerous human disorders. Development of high-throughput analytical methods such as liquid chromatography coupled on-line to mass spectrometry provides the possibility to address epilipidome diversity in complex biological samples. However, the main bottleneck of redox lipidomics, the branch of lipidomics dealing with the characterization of oxidized lipids, remains the lack of optimal computational tools for robust, accurate and specific identification of already discovered and yet unknown modified lipids. Here we discuss the main principles of high-throughput identification of lipids and their modified forms and review the main software tools currently available in redox lipidomics. Different levels of confidence for software assisted identification of redox lipidome are defined and necessary steps toward optimal computational solutions are proposed.


Assuntos
Cardiolipinas/análise , Ceramidas/análise , Ácidos Graxos não Esterificados/análise , Glicerofosfolipídeos/análise , Lipidômica/tendências , Lisofosfolipídeos/análise , Triglicerídeos/análise , Animais , Cardiolipinas/química , Cardiolipinas/metabolismo , Ceramidas/química , Ceramidas/metabolismo , Cromatografia Líquida , Simulação por Computador , Ácidos Graxos não Esterificados/química , Ácidos Graxos não Esterificados/metabolismo , Glicerofosfolipídeos/química , Glicerofosfolipídeos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Lipidômica/métodos , Lipidômica/estatística & dados numéricos , Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Modelos Químicos , Oxirredução , Software , Espectrometria de Massas em Tandem , Triglicerídeos/química , Triglicerídeos/metabolismo
14.
Free Radic Biol Med ; 144: 156-166, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31212065

RESUMO

Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature.


Assuntos
Ar/análise , Ensaio de Proficiência Laboratorial/normas , Fosfatidilcolinas/isolamento & purificação , Terminologia como Assunto , Cromatografia de Fase Reversa , Europa (Continente) , Humanos , Peroxidação de Lipídeos , Variações Dependentes do Observador , Fosfatidilcolinas/química , Fosfatidilcolinas/classificação , Análise de Componente Principal , Reprodutibilidade dos Testes , Soluções , Espectrometria de Massas em Tandem
15.
Redox Biol ; 20: 467-482, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30466060

RESUMO

Ultraviolet light is the dominant environmental oxidative skin stressor and a major skin aging factor. We studied which oxidized phospholipid (OxPL) mediators would be generated in primary human keratinocytes (KC) upon exposure to ultraviolet A light (UVA) and investigated the contribution of OxPL to UVA responses. Mass spectrometric analysis immediately or 24 h post UV stress revealed significant changes in abundance of 173 and 84 lipid species, respectively. We identified known and novel lipid species including known bioactive and also potentially reactive carbonyl containing species. We found indication for selective metabolism and degradation of selected reactive lipids. Exposure to both UVA and to in vitro UVA - oxidized phospholipids activated, on transcriptome and proteome level, NRF2/antioxidant response signaling, lipid metabolizing enzyme expression and unfolded protein response (UPR) signaling. We identified NUPR1 as an upstream regulator of UVA/OxPL transcriptional stress responses and found this protein to be expressed in the epidermis. Silencing of NUPR1 resulted in augmented expression of antioxidant and lipid detoxification genes and disturbed the cell cycle, making it a potential key factor in skin reactive oxygen species (ROS) responses intimately involved in aging and pathology.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Proteínas de Neoplasias/genética , Oxirredução/efeitos da radiação , Fosfolipídeos/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/efeitos da radiação , Raios Ultravioleta , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Metaboloma , Metabolômica/métodos , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Transcriptoma
16.
Sci Rep ; 7(1): 15138, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29123162

RESUMO

Oxidized phospholipids (oxPLs) have been recently recognized as important mediators of various and often controversial cellular functions and stress responses. Due to the low concentrations in vivo, oxPL detection is mostly performed by targeted mass spectrometry. Although significantly improving the sensitivity, this approach does not provide a comprehensive view on oxPLs required for understanding oxPL functional activities. While capable of providing information on the diversity of oxPLs, the main challenge of untargeted lipidomics is the absence of bioinformatics tools to support high-throughput identification of previously unconsidered, oxidized lipids. Here, we present LPPtiger, an open-source software tool for oxPL identification from data-dependent LC-MS datasets. LPPtiger combines three unique algorithms to predict oxidized lipidome, generate oxPL spectra libraries, and identify oxPLs from tandem MS data using parallel processing and a multi-scoring identification workflow.

18.
Redox Biol ; 5: 195-204, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25974625

RESUMO

Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated.


Assuntos
Cumarínicos/química , Hidrazinas/química , Fosfolipídeos/metabolismo , Proteínas/metabolismo , Animais , Células Cultivadas , Cromatografia em Camada Fina , Corantes Fluorescentes/química , Humanos , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Fosfolipídeos/análise , Fosfolipídeos/química , Carbonilação Proteica , Proteínas/química , Ratos , Espectrometria de Massas em Tandem
19.
Redox Biol ; 4: 158-68, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25560244

RESUMO

Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment.


Assuntos
Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Peroxidação de Lipídeos , Fígado/metabolismo , Aldeídos/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Fígado Gorduroso/patologia , Células Hep G2 , Hepatócitos/patologia , Humanos , Cetonas/metabolismo , Fígado/patologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
20.
Free Radic Biol Med ; 75 Suppl 1: S15, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26461293

RESUMO

Within the wide range of oxidative modifications, "carbonylation" formed by the incorporation of aldehyde/keto groups, is commonly studied due to its role in cell physiology and as prospective biomarkers for numerous disorders. Despite close biochemical and physiological links between protein and lipid carbonylation, these two types of modifications are rarely addressed simultaneously in a single study. In nitrosative stress cell model we investigated levels of protein and lipid carbonylation and addressed the main modified species by combining LC-MS, biochemical, and microscopy studies. The influence of nitrosative stress on carbonylation of proteins and lipids was investigated for primary cardiomyocytes treated with SIN-1 for different time intervals. Lipid carbonylation was quantified by RPC-ESI-MS/MS. The results demonstrate dynamic generation, degradation and adduct formations of 25 different species including alkanals, alkenals, alkadienals, alkatrienals and oxo-carboxylic acids. Several new PL-bound aldehydes were present exclusively after a long incubation period. Carbonylated proteins were identified after aldehyde reactive probe derivatization, affinity enrichment and RPC-ESI-MS/MS. More than 200 proteins were identified and evaluated by systems biology to deduce the biological significance of the protein modifications. The protein carbonylation degree was verified using oxyblot and correlated with changes in 20S/26S proteasome activities. Furthermore, a new fluorescence microscopy based technique to stain carbonylated biomolecules was developed and compared with conventional DNPH-based immunocytochemistry. Subcellular localization of carbonylated species was investigated using mitochondrial and ER-specific co-localization experiments. Thus, the combination of lipidomics, proteomics, biochemical techniques, and microscopy imaging revealed a complex molecular pattern of "carbonylation stress" in the studied nitrosative stress cell model.

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