RESUMO
Mass spectrometry (MS) as an analytical technique enables the identification and quantitative determination of proteins, metabolites, or lipids in a studied sample. However, this method has limitations regarding the number of molecules that can be identified at a given time. To increase the number of identifications, the application of ion mobility spectrometry (IMS) can be employed. This technique allows the separation of ions based on their mobility while traversing the analyser in a gradient of an electromagnetic field and opposing gas pressure. The separation is performed in conjunction with MS analysis, adding another dimension to the analysis, resulting in a significant improvement in the number of identified compounds and a reduction in noise. Alternatively, while maintaining the same number of identifications, analysis can be performed in a shorter time period. It is crucial to pay special attention to the type of IMS analyser used, as its specific implementation dictates further stages of analysis and ion detection capabilities.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Metabolômica/métodos , Humanos , Íons/análise , Proteínas/análise , MultiômicaRESUMO
Mass spectrometry is an important tool in proteomic, metabolomic and lipidomic analysis. To fully use its potential, it is crucial to select and configure the appropriate analytical approach. For untargeted research, there are two main strategies available: data-dependent analysis (DDA) and data-independent analysis (DIA). Both methods differ in the way the analysis is carried out and in the degree of coverage of the obtained data, which is why each of them can be used in various types of research. The DDA method is based on continuous scanning of the analyzed ions, as a result of which the precursors with the highest intensity are fragmented in the MS2 mode. On the other hand, DIA, due to the use of combined ranges of precursor ion isolation, allows for a deeper analysis of the analyzed compounds. Both approaches also have modifications that improve their operation and enable obtaining more valuable data. Methods combining both techniques are also appearing on the horizon, such as DDIA, which uses the advantages of both methods, opening new analytical possibilities.
Assuntos
Espectrometria de Massas , Espectrometria de Massas/métodos , Proteômica/métodos , Metabolômica/métodos , Humanos , AnimaisRESUMO
A process of plant recovery after drought cessation is a complex trait which has not been fully recognized. The most important organ associated with this phenomenon in monocots, including forage grasses, is the crown tissue located between shoots and roots. The crown tissue is a meristematic crossroads for metabolites and other compounds between these two plant organs. Here, for the first time, we present a metabolomic and lipidomic study focused on the crown tissue under drought and recovery in forage grasses, important for agriculture in European temperate regions. The plant materials involve high (HDT) and low drought-tolerant (LDT) genotypes of Festuca arundinacea, and Lolium multiflorum/F. arundinacea introgression forms. The obtained results clearly demonstrated that remodeling patterns of the primary metabolome and lipidome in the crown under drought and recovery were different between HDT and LDT plants. Furthermore, HDT plants accumulated higher contents of primary metabolites under drought in the crown tissue, especially carbohydrates which could function as osmoprotectants and storage materials. On the other hand, LDT plants characterized by higher membranes damage under drought, simultaneously accumulated membrane phospholipids in the crown and possessed the capacity to recover their metabolic functions after stress cessation to the levels observed in HDT plants.
Assuntos
Resistência à Seca , Festuca , Lolium , Resistência à Seca/genética , Secas , Festuca/genética , Festuca/metabolismo , Lolium/genética , Lolium/metabolismo , GenótipoRESUMO
Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.
Assuntos
Carbono/metabolismo , Nitrogênio/metabolismo , Pisum sativum/fisiologia , Adaptação Fisiológica , Transporte Biológico , Secas , Genótipo , Ácido Oleico/metabolismo , Pisum sativum/anatomia & histologia , Pisum sativum/genética , Floema/anatomia & histologia , Floema/genética , Floema/fisiologia , Exsudatos de Plantas , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Estresse Fisiológico , Água/fisiologiaRESUMO
The energy-dense western diet significantly increases the risk of obesity, type 2 diabetes, cardiovascular episodes, stroke, and cancer. Recently more attention has been paid to the contribution of an unhealthy lifestyle on the development of central nervous system disorders. Exposure to long-lasting stress is one of the key lifestyle modifications associated with the increased prevalence of obesity and metabolic diseases. The main goal of the present study was to verify the hypothesis that exposure to chronic stress modifies alterations in the brain proteome induced by the western diet. Female adult rats were fed with the prepared chow reproducing the human western diet and/or subjected to chronic stress induced by social instability for 6 weeks. A control group of lean rats were fed with a standard diet. Being fed with the western diet resulted in an obese phenotype and induced changes in the serum metabolic parameters. The combination of the western diet and chronic stress exposure induced more profound changes in the rat cerebrocortical proteome profile than each of these factors individually. The down-regulation of proteins involved in neurotransmitter secretion (Rph3a, Snap25, Syn1) as well as in learning and memory processes (Map1a, Snap25, Tnr) were identified, while increased expression was detected for 14-3-3 protein gamma (Ywhag) engaged in the modulation of the insulin-signaling cascade in the brain. An analysis of the rat brain proteome reveals important changes that indicate that a combination of the western diet and stress exposure may lead to impairments of neuronal function and signaling.
Assuntos
Diabetes Mellitus Tipo 2 , Dieta Ocidental , Animais , Dieta Hiperlipídica , Dieta Ocidental/efeitos adversos , Feminino , Insulina , Obesidade/etiologia , Obesidade/metabolismo , Proteoma/metabolismo , RatosRESUMO
The aim of this study was to demonstrate the metabolic profile of post-culture medium as an expression of cell suspension metabolic activity of the tree fern Cyathea delgadii Sternb. The molecular profile of the tree fern's cell culture has been never described, according to our knowledge. The cell suspension was established using ½ MS medium supplemented with various concentrations of 2,4-D and BAP. The optimal concentrations were 2.0 mg·L-1 and 0.2 mg·L-1, respectively. The cell suspension initially showed an organized system of cell division and later unorganized cell proliferation. LC-MS and GC-MS were used to identify the chemical composition of the post-culture medium. The LC-MS analysis results suggested that the color of liquid medium could be due to the presence of flavonoid derivatives, as this group of compounds was represented by eight compounds. After GC-MS analysis based on retention indexes and thanks to mass spectra comparison, 130 natural products were recognized, belonging to various classes of primary and secondary metabolites.
Assuntos
Produtos Biológicos , Gleiquênias , Traqueófitas , Ácido 2,4-Diclorofenoxiacético , Cromatografia Líquida/métodos , Flavonoides/análiseRESUMO
A progressive loss of functional nephrons defines chronic kidney disease (CKD). Complications related to cardiovascular disease (CVD) are the principal causes of mortality in CKD; however, the acceleration of CVD in CKD remains unresolved. Our study used a complementary proteomic approach to assess mild and advanced CKD patients with different atherosclerosis stages and two groups of patients with different classical CVD progression but without renal dysfunction. We utilized a label-free approach based on LC-MS/MS and functional bioinformatic analyses to profile CKD and CVD leukocyte proteins. We revealed dysregulation of proteins involved in different phases of leukocytes' diapedesis process that is very pronounced in CKD's advanced stage. We also showed an upregulation of apoptosis-related proteins in CKD as compared to CVD. The differential abundance of selected proteins was validated by multiple reaction monitoring, ELISA, Western blotting, and at the mRNA level by ddPCR. An increased rate of apoptosis was then functionally confirmed on the cellular level. Hence, we suggest that the disturbances in leukocyte extravasation proteins may alter cell integrity and trigger cell death, as demonstrated by flow cytometry and microscopy analyses. Our proteomics data set has been deposited to the ProteomeXchange Consortium via the PRIDE repository with the data set identifier PXD018596.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Insuficiência Renal Crônica , Aterosclerose/genética , Cromatografia Líquida , Humanos , Integrinas , Leucócitos , Proteômica , Insuficiência Renal Crônica/genética , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: Obesity is a multidimensional condition that is treatable by the restoration of a lean phenotype; however, some obesity-related outcomes may persist after weight normalization. Among the organs of the human body, the brain possesses a relatively low regenerative capacity and could retain perturbations established as a result of developmental obesity. Calorie restriction (CR) or a restricted ketogenic diet (KD) are successfully used as weight loss approaches, but their impact on obesity-related effects in the brain have not been previously evaluated. METHODS: We performed a series of experiments in a rat model of developmental obesity induced by a 12-week cafeteria diet, followed by CR to implement weight loss. First, we assessed the impact of obesity on neurogenesis (BrdU incorporation into the hippocampus), cognitive function (water maze), and concomitant changes in hippocampal protein expression (GC/MS-MS, western blot). Next, we repeated these experiments in a rat model of weight loss induced by CR. We also measured mitochondrial enzyme activity in rats after weight loss during the fed or fasting state. This study was extended by additional experiments with restricted KD used as a weight loss approach in order to compare the efficacy of two different nutritional interventions used in the treatment of obesity on hippocampal functions. By using a modified version of the water maze we evaluated cognitive abilities in rats subjected to weight loss by CR or a restricted KD. RESULTS: In this study, obesity affected metabolic processes, upregulated hippocampal NF-κB, and induced proteomic differences which were associated with impaired cognition and neurogenesis. Weight loss improved neurogenesis and enhanced cognition. While the expression pattern of some proteins persisted after weight loss, most of the changes appeared de novo revealing metabolic adjustment by overactivation of citrate synthase and downregulation of ATP synthase. As a consequence of fasting, the activity of these enzymes indicated hippocampal adaptation to negative energy balance during the weight loss phase of CR. Moreover, the effects on cognitive abilities measured after weight loss were negatively correlated with the animal weight measured at the final stage of weight gain. This was alleviated by KD, which improved cognition when used as a weight loss approach. CONCLUSIONS: The study shows that cognition and mitochondrial metabolism in the hippocampus are affected by CR- or KD-induced weight loss.
Assuntos
Proteômica , Redução de Peso , Animais , Restrição Calórica , Hipocampo , Obesidade/complicações , RatosRESUMO
The commonly observed increased heavy metal tolerance of ectomycorrhized plants is usually linked with the protective role of the fungal hyphae covering colonized plant root tips. However, the molecular tolerance mechanisms in heavy metal stressed low-colonized ectormyocrrhizal plants characterized by an ectomycorrhiza-triggered increases in growth are unknown. Here, we examined Populus × canescens microcuttings inoculated with the Paxillus involutus isolate, which triggered an increase in poplar growth despite successful colonization of only 1.9% ± 0.8 of root tips. The analyzed plants, lacking a mantle-a protective fungal biofilter-were grown for 6 weeks in agar medium enriched with 0.75 mM Pb(NO3)2. In minimally colonized 'bare' roots, the proteome response to Pb was similar to that in noninoculated plants (e.g., higher abundances of PM- and V-type H+ ATPases and lower abundance of ribosomal proteins). However, the more intensive activation of molecular processes leading to Pb sequestration or redirection of the root metabolic flux into amino acid and Pb chelate (phenolics and citrate) biosynthesis coexisted with lower Pb uptake compared to that in controls. The molecular Pb response of inoculated roots was more intense and effective than that of noninoculated roots in poplars.
Assuntos
Basidiomycota/fisiologia , Chumbo/farmacologia , Doenças das Plantas/imunologia , Raízes de Plantas/metabolismo , Populus/metabolismo , Proteoma/metabolismo , Estresse Fisiológico , Micorrizas/efeitos dos fármacos , Micorrizas/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Populus/efeitos dos fármacos , Populus/microbiologia , Proteoma/análiseRESUMO
Ectomycorrhizae (ECMs) are a highly context-dependent interactions that are not always beneficial for the plant host, sometimes leading to a decrease in plant growth. However, the molecular status of these plants remains unknown. We studied Populus × canescens microcuttings characterized by impaired growth in response to colonization by a Paxillus involutus strain via integrative proteomics-metabolomics analyses. The analysed strain was characterized by low compatibility and formed only mantles, not a Hartig net, in the majority of root tips. The increased abundance of photosynthetic proteins and foliar carbohydrates co-occurred with signals of intensified resource exchange via the stems of colonized plants. In the roots, intensified C metabolism resulted in the biosynthesis of secondary C compounds unavailable to the fungal partner but also C skeletons necessary to increase insufficient N uptake from the hyphae. The stress response was also detected in colonized plants but was similar to that reported previously during mutualistic ECM interactions. In colonized poplar plants, mechanisms to prevent imbalanced C/N trade-offs were activated. Root metabolism strongly depended on features of the whole plant, especially the foliar C/N budget. However, despite ECM-triggered growth impairment and the foliar nutrient status, the fungal partner was recognized to be a symbiotic partner.
Assuntos
Basidiomycota , Micorrizas , Populus , Metabolômica , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Populus/crescimento & desenvolvimento , Populus/metabolismo , Populus/microbiologia , Proteômica , SimbioseRESUMO
Drought is a major abiotic stress that negatively influences crop yield. Breeding strategies for improved drought resistance require an improved knowledge of plant drought responses. We therefore applied drought to barley recombinant inbred lines and their parental genotypes shortly before tillering. A large-scale proteomic analysis of leaf and root tissue revealed proteins that respond to drought in a genotype-specific manner. Of these, Rubisco activase in chloroplast, luminal binding protein in endoplasmic reticulum, phosphoglycerate mutase, glutathione S-transferase, heat shock proteins and enzymes involved in phenylpropanoid biosynthesis showed strong genotype×environment interactions. These data were subjected to genetic linkage analysis and the identification of proteomic QTLs that have potential value in marker-assisted breeding programs.
Assuntos
Hordeum/metabolismo , Proteínas de Plantas/genética , Proteoma/genética , Locos de Características Quantitativas , Secas , Genótipo , Hordeum/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Proteoma/metabolismo , ProteômicaRESUMO
During ectomycorrhizal symbioses, up to 30% of the carbon produced in leaves may be translocated to the fungal partner. Given that the leaf response to root colonization is largely unknown, we performed a leaf proteome analysis of Populus × canescens inoculated in vitro with two isolates of Paxillus involutus significantly differing in root colonization rates (65 ± 7% vs 14 ± 7%), together with plant growth and leaf biochemistry analyses to determine the response of plant leaves to ectomycorrhizal root colonization. The isolate that more efficiently colonized roots (isolate H) affected 9.1% of the leaf proteome compared with control plants. Simultaneously, ectomycorrhiza in isolate H-inoculated plants led to improved plant growth and an increased abundance of leaf proteins involved in protein turnover, stress response, carbohydrate metabolism, and photosynthesis. The protein increment was also correlated with increases in chlorophyll, foliar carbon, and carbohydrate contents. Although inoculation of P. × canescens roots with the other P. involutus isolate (isolate L, characterized by a low root colonization ratio) affected 6.8% of the leaf proteome compared with control plants, most proteins were downregulated. The proteomic signals of increased carbohydrate biosynthesis were not detected, and carbohydrate, carbon, and leaf pigment levels and plant biomass did not differ from the noninoculated plants. Our results revealed that the upregulation of the photosynthetic protein abundance and levels of leaf carbohydrate are positively related to rates of root colonization. Upregulation of photosynthetic proteins, chlorophyll, and leaf carbohydrate levels in ectomycorrhizal plants was positively related to root colonization rates and resulted in increased carbon translocation and sequestration underground.
Assuntos
Basidiomycota/fisiologia , Micorrizas/fisiologia , Populus/microbiologia , Proteoma , Fotossíntese/genética , Folhas de Planta/química , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologiaRESUMO
Ischemic stroke induces brain injury via thrombotic or embolic mechanisms involving large or small vessels. Cystathionine ß-synthase deficiency (CBS), an inborn error of metabolism, is associated with vascular thromboembolism, the major cause of morbidity and mortality in affected patients. Because thromboembolism involves the brain vasculature in these patients, we hypothesize that CBS deficiency and ischemic stroke have similar molecular phenotypes. We used label-free mass spectrometry for quantification of changes in serum proteomes in CBS-deficient patients (n = 10) and gender/age-matched unaffected controls (n = 14), as well as in patients with cardioembolic (n = 17), large-vessel (n = 26), or lacunar (n = 25) ischemic stroke subtype. In CBS-deficient patients, 40 differentially expressed serum proteins were identified, of which 18 were associated with elevated homocysteine (Hcy) and 22 were Hcy-independent. We also identified Hcy-independent differentially expressed serum proteins in ischemic stroke patients, some of which were unique to a specific subtype: 10 of 32 for cardioembolic vs. large-vessel, six of 33 for cardioembolic vs. lacunar, and six of 23 for large-vessel vs. lacunar. There were significant overlaps between proteins affected by CBS deficiency and ischemic stroke, particularly the cardioembolic subtype, similar to protein overlaps between ischemic stroke subtypes. Top molecular pathways affected by CBS deficiency and ischemic stroke subtypes included acute phase response signaling and coagulation system. Similar molecular networks centering on NFκB were affected by CBS deficiency and stroke subtypes. These findings suggest common mechanisms involved in the pathologies of CBS deficiency and ischemic stroke subtypes.
Assuntos
Biomarcadores/sangue , Isquemia Encefálica/complicações , Cistationina beta-Sintase/deficiência , Proteoma , Proteômica , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/etiologia , Adulto , Biologia Computacional/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Transdução de SinaisRESUMO
A wide range of compounds that occur in the genus Hypericum are listed as effective drugs of natural origin. The main biological activities of several Hypericum representatives are due to the presence of naphthodianthrones, phloroglucinols, and other diverse groups of secondary metabolites that synergistically contribute to their therapeutic effects. The regulation of biosynthesis of hypericin as the key bioactive naphthodianthrone remains uncertain. Here, we present liquid chromatography mass spectrometry-based phenotyping of 17 Hypericum species, the results of which suggest an important role for skyrin and its derivatives in the polyketide pathway that leads to hypericin formation. Moreover, we report for the first time the presence of new metabolites in the genus Hypericum that are related to classes of anthraquinones, their derivatives, and phloroglucinols. As skyrin and other species of anthraquinones are rarely found in higher plants but frequently occur in fungal microorganisms, the obtained results suggest that further research on the synthesis pathways of hypericin and the role of anthraquinone derivatives in plant metabolism should be carried out. The fact that these compounds are commonly synthesized in endophytic fungi and perhaps there is some similarity in the metabolic pathways between these organisms should also be investigated.
Assuntos
Antraquinonas/química , Emodina/química , Hypericum , Perileno/análogos & derivados , Metabolismo Secundário , Antracenos , Cromatografia Líquida , Espectrometria de Massas , Perileno/química , Fenótipo , Folhas de Planta/químicaRESUMO
The aim of this study was to investigate the effect of exogenous nitric oxide (NO), i.e., S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the metabolic status of Pisum sativum L. cv. Cysterski leaves infested by Acyrthosiphon pisum Harris, population demographic parameters and A. pisum feeding activity. A reduction in the level of semiquinone radicals in pea seedling leaves pretreated with exogenous NO occurred 24 h after A. pisum infestation, which was earlier than in non-pretreated leaves. A decrease in the level of O2â¢- was observed in leaves pretreated with GSNO and infested by aphids at 48 and 72 h post-infestation (hpi). Directly after the pretreatment with GSNO, an increase in the level of metal ions was recorded. NO considerably induced the relative mRNA levels for phenylalanine ammonia-lyase in 24-h leaves pretreated with NO donors, both non-infested and infested. NO stimulated the accumulation of pisatin in leaves until 24 h. The Electrical Penetration Graph revealed a reduction in the feeding activity of the pea aphid on leaves pretreated with NO. The present study showed that foliar application of NO donors induced sequentially defense reactions of pea against A. pisum and had a deterrent effect on aphid feeding and limited the population growth rate.
Assuntos
Afídeos/fisiologia , Interações Hospedeiro-Parasita , Infestações por Piolhos , Óxido Nítrico/biossíntese , Pisum sativum/fisiologia , Pisum sativum/parasitologia , Animais , Afídeos/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Benzoquinonas/metabolismo , Resistência à Doença , Flavonoides/metabolismo , Glicosídeos/metabolismo , Ferro/química , Ferro/metabolismo , Manganês/química , Manganês/metabolismo , Óxido Nítrico/farmacologia , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Pterocarpanos/metabolismo , Superóxidos/metabolismo , beta-Glucosidase/metabolismoRESUMO
The aim of this study was to investigate the effect of an abiotic factor, i.e., lead at various concentrations (low causing a hormesis effect and causing high toxicity effects), on the generation of signalling molecules in pea (Pisum sativum L. cv. Cysterski) seedlings and then during infestation by the pea aphid (Acyrthosiphon pisum Harris). The second objective was to verify whether the presence of lead in pea seedling organs and induction of signalling pathways dependent on the concentration of this metal trigger defense responses to A. pisum. Therefore, the profile of flavonoids and expression levels of genes encoding enzymes of the flavonoid biosynthesis pathway (phenylalanine ammonialyase and chalcone synthase) were determined. A significant accumulation of total salicylic acid (TSA) and abscisic acid (ABA) was recorded in the roots and leaves of pea seedlings growing on lead-supplemented medium and next during infestation by aphids. Increased generation of these phytohormones strongly enhanced the biosynthesis of flavonoids, including a phytoalexin, pisatin. This research provides insights into the cross-talk between the abiotic (lead) and biotic factor (aphid infestation) on the level of the generation of signalling molecules and their role in the induction of flavonoid biosynthesis.
Assuntos
Afídeos , Flavonoides/metabolismo , Chumbo/farmacologia , Pisum sativum/fisiologia , Pisum sativum/parasitologia , Plântula/efeitos dos fármacos , Plântula/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interações Hospedeiro-Parasita/genética , Metaboloma , Metabolômica/métodos , Doenças das Plantas/parasitologia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Pterocarpanos/metabolismo , Ácido Salicílico/metabolismo , Plântula/crescimento & desenvolvimentoRESUMO
Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznan, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznan.
Assuntos
Proteômica , Animais , Biomarcadores/metabolismo , Europa Oriental , Humanos , Proteoma/metabolismo , Biologia de SistemasRESUMO
Currently, research interest is increasing in horse milk composition and its effect on human health. Despite previously published studies describing the presence of intra- and interbreed variability of equine milk components, no investigations have focused on the genetic background of this variation. Among horse caseins and the genes encoding them, least is known about the structure and expression of the α-S2 casein gene, CSN1S2. Herein, based on direct sequencing of the equine CSN1S2 coding sequence, we describe the presence of 51-bp insertion-deletion (in/del) polymorphism, which significantly changes the protein sequence (lack or presence of 17-amino acid serine-rich peptide). Bioinformatic analysis revealed that the observed in/del polymorphism spanned exactly 2 exons; therefore, we hypothesized that we were observing different CSN1S2 splicing isoforms. However, further investigation indicated that the detected sequence variation was caused by a large (1.3-kb) deletion in the genomic DNA. We found that the polymorphic forms (A, longer; B, shorter; KP658381 and KP658382 GenBank records, respectively) were unevenly distributed among different horse breeds (the highest frequency of variant B was observed in coldblood horses and Haflingers). We propose that the analyzed polymorphism is associated with CSN1S2 expression level (the highest expression was recorded for individuals carrying the BB genotype), which was much more pronounced for milk CSN1S2 protein content than for relative transcript abundance (measured in milk somatic cells). Our results provide insight into the equine CSN1S2 structure and lay a foundation for further functional analyses regarding, for example, allergenicity or physiochemical properties of the observed CSN1S2 variants.
Assuntos
Caseínas/genética , Variação Genética/genética , Cavalos/genética , Proteômica , Transcriptoma/genética , Sequência de Aminoácidos , Animais , Cruzamento , Caseínas/química , DNA/química , DNA/genética , Deleção de Genes , Genótipo , Leite/química , Proteínas do Leite/análise , Dados de Sequência Molecular , Fases de Leitura Aberta , Polimorfismo Genético/genética , RNA Mensageiro/análiseRESUMO
The major cause of mortality in patients with chronic kidney disease (CKD) is atherosclerosis related to traditional and non-traditional risk factors. However, the understanding of the molecular specificity that distinguishes the risk factors for classical cardiovascular disease (CVD) and CKD-related atherosclerosis (CKD-A) is far from complete. In this study we investigated the disease-related differences in the proteomes of patients with atherosclerosis related and non-related to CKD. Plasma collected from patients in various stages of CKD, CVD patients without symptoms of kidney dysfunction, and healthy volunteers (HVs), were analyzed by a coupled label-free and mass spectrometry approach. Dysregulated proteins were confirmed by an enzyme-linked immunosorbent assay (ELISA). All proteomic data were correlated with kidney disease development and were subjected to bioinformatics analysis. One hundred sixty-two differentially expressed proteins were identified. By directly comparing the plasma proteomes from HVs, CKD, and CVD patients in one study, we demonstrated that proteins involved in inflammation, blood coagulation, oxidative stress, vascular damage, and calcification process exhibited greater alterations in patients with atherosclerosis related with CKD. These data indicate that the above nontraditional risk factors are strongly specific for CKD-A and appear to be less essential for the development of "classical" CVD.
Assuntos
Aterosclerose/etiologia , Aterosclerose/fisiopatologia , Proteômica , Insuficiência Renal Crônica/complicações , Aterosclerose/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Humanos , Osteopontina/metabolismo , Peroxirredoxinas/metabolismo , Análise de Componente Principal , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Fatores de Risco , Espectrometria de Massas em Tandem , alfa-2-Glicoproteína-HS/metabolismoRESUMO
Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material including flavonoid glycoconjugates. Resolution of the applied mass spectrometers plays an important role in structural studies of mixtures of the target compounds isolated from biological material. High-resolution analyzers allow obtaining information about elemental composition of the analyzed compounds. Application of various mass spectrometric techniques, including different systems of ionization, analysis of both positive and negative ions of flavonoids, fragmentation of the protonated/deprotonated molecules and in some cases addition of metal ions to the studied compounds before ionization and fragmentation, may improve structural characterization of natural products. In our review we present different strategies allowing structural characterization of positional isomers and isobaric compounds existing in class of flavonoid glycoconjugates and their derivatives, which are synthetized in plants and are important components of the human food and drugs as well as animal feed.