RESUMO
It is undeniable that as people get older, they become progressively more susceptible to neurodegenerative illnesses such as Alzheimer's disease (AD). Memory loss is a prominent symptom of this condition and can be exacerbated by uneven levels of certain metals. This study used inductively coupled plasma mass spectrometry (ICP-MS) to examine the levels of metals in the blood plasma, frontal cortex, and hippocampus of Wistar rats with AD induced by streptozotocin (STZ). It also tested the effects of the antioxidant hydroxytyrosol (HT) on metal levels. The Barnes maze behavior test was used, and the STZ group showed less certainty and greater distance when exploring the Barnes maze than the control group. The results also indicated that the control group and the STZ + HT group exhibited enhanced learning curves during the Barnes maze training as compared to the STZ group. The ICP-MS analysis showed that the STZ group had lower levels of cobalt in their blood plasma than the control group, while the calcium levels in the frontal cortex of the STZ + HT group were higher than in the control group. The most important finding was that copper levels in the frontal cortex from STZ-treated animals were higher than in the control group, and that the STZ + HT group returned to equivalent levels to the control group. The antioxidant HT can restore copper levels to their basal physiological state. This finding may help explain HT's potential beneficial effect in AD-patients.
Assuntos
Doença de Alzheimer , Humanos , Ratos , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/induzido quimicamente , Ratos Wistar , Antioxidantes/efeitos adversos , Cobre/farmacologia , Modelos Animais de Doenças , Hipocampo , Estreptozocina/efeitos adversos , Aprendizagem em LabirintoRESUMO
Results obtained from rat studies indicate that, even at low concentrations, mercurial species cause harmful effects on the kidneys, by inducing the nephrotic oxidative stress response. In the present work, Hg-associated proteins were identified as possible mercury-exposure biomarkers in rat kidneys exposed to low mercury chloride concentrations for 30 days (Hg-30) and 60 days (Hg-60), using metalloproteomic strategies. The renal proteomic profile was fractioned by two-dimensional electrophoresis and the mercury determinations in kidney samples, protein pellets and protein spots were performed using graphite furnace atomic absorption spectrometry. The characterization of Hg-associated protein spots and the analysis of differentially expressed proteins were performed by liquid chromatography, coupled with tandem mass spectrometry. Eleven Hg-associated protein spots with a concentration range of 79 ± 1 to 750 ± 9 mg kg-1 in the Hg-60 group were identified. The characterization and expression analyses allowed the identification of 53 proteins that were expressed only in the Hg-60 group, 13 "upregulated" proteins (p > 0.95) and 47 "downregulated" proteins (p < 0.05). Actin isoforms and hemoglobin subunits were identified in protein spots of the Hg-60 group, with mercury concentrations in the range of 138 to 750 mg kg-1, which qualifies these proteins as potential mercury-exposure biomarkers.
Assuntos
Desequilíbrio Ácido-Base , Mercúrio , Animais , Ratos , Proteínas de Transporte , Cloretos , Proteômica , Cloreto de Mercúrio/toxicidade , Mercúrio/toxicidade , BiomarcadoresRESUMO
Metalloproteomics is an innovative methodology for identifying of protein-associated mercury. Thus, we analyzed the muscle proteome of Arapaima gigas (pirarucu), collected in the Madeira River of the Brazilian Amazon, to identify protein-associated mercury, with the aim of identifying possible mercury biomarkers in fish muscle tissue. After obtaining the protein pellet, we conducted two-dimensional electrophoresis (2D PAGE) to fractionate the muscle proteome. Total mercury in muscle tissue and protein pellets and mapping of mercury content in protein spots of the 2D PAGE gels was determined using graphite furnace atomic absorption spectrometry (GFAAS). The protein-associated mercury identification was performed using liquid chromatography coupled with sequence mass spectrometry (LCâMS/MS). Total mercury determinations by GFAAS indicated concentrations on the order of 153 ± 1.90 mg kg-1 and 142 ± 1.50 mg kg-1 (total precipitation of protein fraction) and 139 ± 1.45 mg kg-1 (fractional precipitation of protein fraction) in muscle tissue and protein pellets, respectively. Mercury concentrations in the range of 48 ± 0.90 to 165 ± 3.00 mg kg-1 were found in twelve protein spots. Among the 2D PAGE protein spots, eleven Hg-binding proteins were identified using LCâMS/MS, which showed characteristics of mercury exposure biomarkers for important metabolic functions, such as five parvalbumin isoforms, triosephosphate isomerase, cofilin 2 (muscle), and fructose-bisphosphate aldolases.
Assuntos
Mercúrio , Poluentes Químicos da Água , Animais , Biomarcadores/metabolismo , Brasil , Cromatografia Líquida , Monitoramento Ambiental , Peixes/metabolismo , Mercúrio/análise , Músculos/química , Proteoma , Espectrometria de Massas em Tandem , Poluentes Químicos da Água/análiseRESUMO
Five newly synthetized fac-Re(I) tricarbonyl compounds were explored as prospective antitrypanosomal agents. The biological activity of the whole series was evaluated preliminarily against the epimastigote form of Trypanosoma cruzi. All compounds showed activity against epimastigotes with IC50 values in the low micromolar range. The most active compound [fac-Re(I)(CO)3(tmp)(CTZ)](PF6), with CTZ = clotrimazole and tmp = 3,4,7,8-tetramethyl-1,10-phenantroline, showed good selectivity towards the parasites and thus was selected to carry out further metallomic studies. For this task, a newly bioanalytical method based on microwave plasma atomic emission spectrometry (MP-AES) was developed and validated. The accuracy of the method was ensured by testing a certified reference material. Results of rhenium elemental analysis by MP-AES agreed with the proposed formula of the studied compounds, contributing to the overall validation of the method, which was then applied to evaluate the percentage of rhenium uptaken by the parasites and the association of the compounds with parasite biomacromolecules. Metallomics results showed low total rhenium percentage uptaken by parasites (â¼1.2%) and preferential accumulation in the soluble proteins fraction (â¼82.8%). Thus, the method based on MP-AES turned out to be an economical and green alternative for metallomics studies involving potential rhenium metallodrugs. Moreover, a comparison against rhenium determination by electrothermal atomic absorption spectrometry (ET-AAS) was included.
Assuntos
Rênio , Micro-Ondas , Plasma , Estudos Prospectivos , Rênio/química , Espectrofotometria Atômica/métodosRESUMO
Potential use of metal complexes in medicine is an area of bioinorganic chemistry that has gained much interest. High-throughput omics approaches can provide in-depth insights into the mechanism of action of new metal-based compounds. Discovering new metallodrugs against Trypanosoma cruzi is an emerging field. Combining metallomics, proteomics, and transcriptomics allows the identification of multiple molecular targets and several parasitic metabolic pathways affected by V(IV), Pt(II), and Pd(II) potential antiparasitic drugs. Specifically, metallomics studies with Pd(II) and Pt(II) analogous compounds show higher parasite uptake of the Pt(II) than Pd(II), and both accumulate similarly in the parasite DNA fraction. Unexpectedly, vanadium did not associate with DNA. The studies reviewed illustrate the use of omics techniques for determining molecular targets of potential therapeutic agents.
Assuntos
Complexos de Coordenação , Tripanossomicidas , Trypanosoma cruzi , Antiparasitários/farmacologia , Complexos de Coordenação/química , DNA , Paládio/química , Tripanossomicidas/química , Tripanossomicidas/farmacologiaRESUMO
Five heteroleptic compounds, [VVO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC50 values of 0.29-3.02 µM. IN ligand and the new [VVO2(IN-H)] complex showed negligible activity. The most active compound [VVO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [VVO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC50 values of [VVO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 µM concentration of [VVO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Oxiquinolina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Vanádio/farmacologia , Animais , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Estrutura Molecular , Oxiquinolina/química , Testes de Sensibilidade Parasitária , Tripanossomicidas/síntese química , Tripanossomicidas/química , Vanádio/químicaRESUMO
Marine bivalves have been widely applied as environmental contamination bioindicators, although studies concerning tropical species are less available compared to temperate climate species. Assessments regarding Perna perna mytilid mussels, in particular, are scarce, even though this is an extremely important species in economic terms in tropical countries, such as Brazil. To this end, Perna perna mytilids were sampled from two tropical bays in Southeastern Brazil, one anthropogenically impacted and one previously considered a reference site for metal contamination. Gill metallothionein (MT), reduced glutathione (GSH), carboxylesterase (CarbE) and lipid peroxidation (LPO) were determined by UV-vis spectrophotometry, and metal and metalloid contents were determined by inductively coupled plasma mass spectrometry (ICP-MS). Metalloprotein metal detoxification routes in heat-stable cellular gill fractions were assessed by size exclusion high performance chromatography (SEC-HPLC) coupled to an ICP-MS. Several associations between metals and oxidative stress endpoints were observed at all four sampling sites through a Principal Component Analysis. As, Cd, Ni and Se contents, in particular, seem to directly affect CarbE activity. MT is implicated in playing a dual role in both metal detoxification and radical oxygen species scavenging. Differential SEC-HPLC-ICP-MS metal-binding profiles, and, thus, detoxification mechanisms, were observed, with probable As-, Cu- and Ni-GSH complexation and binding to low molecular weight proteins. Perna perna mussels were proven adequate tropical bioindicators, and further monitoring efforts are recommended, due to lack of data regarding biochemical metal effects in tropical species. Integrated assessments, as performed herein demonstrate, are invaluable in evaluating contaminated aquatic environments, resulting in more accurate ecological risk assessments.
Assuntos
Metais/toxicidade , Perna (Organismo)/fisiologia , Poluentes Químicos da Água/toxicidade , Animais , Baías , Brasil , Monitoramento Ambiental , Brânquias/efeitos dos fármacos , Glutationa/metabolismo , Metaloproteínas/metabolismo , Metalotioneína/metabolismo , Metais/análise , Metais/metabolismo , Perna (Organismo)/efeitos dos fármacos , Alimentos Marinhos/análise , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/metabolismoRESUMO
The development of metallomics techniques has allowed for metallomics analysis of biological systems, enabling a better understanding of the response mechanisms for different stimuli, their relationship to metallic species, and the characterization of biomarkers. In this study, a metallomics analysis of the muscle tissue of Nile tilapia was used to aid the understanding of the molecular mechanisms involved in zinc absorption in this fish species when fed organic and/or inorganic sources of zinc and to identify possible biomarkers for the absorption of this micromineral. To accomplish this, the fish were separated into three groups of 24 g, 74 g, and 85 g initial weights, and each group, respectively, was fed a zinc-free diet (control group, G1), a diet containing zinc found in organic sources (treatment 1, G2), and a diet containing zinc from an inorganic source (treatment 2, G3). Two-dimensional polyacrylamide (2D PAGE) gel electrophoresis was used to separate the proteins of the muscle tissue. Subsequently, the expression profiles of protein spots in the samples where zinc was applied in different concentrations were compared, using the software ImageMaster 2D Platinum version 7.0, to identify proteins that were differentially expressed. The identified proteins were then exposed to atomic absorption spectrometry in a graphite furnace to determine zinc mapping and were subsequently characterized via electrospray ionization tandem mass spectrometry (ESI-MS/MS). The metallomic analysis identified 15 proteins differentially expressed and associated with zinc, leading to the conclusion that three metal-binding proteins presented as possible biomarkers of zinc absorption in fish.
Assuntos
Músculos/química , Zinco/análise , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Peso Corporal , Ciclídeos , Proteínas de Peixes/análise , Proteínas de Peixes/metabolismo , Músculos/metabolismo , Zinco/administração & dosagem , Zinco/metabolismoRESUMO
This study aimed to evaluate the quality of the royal jelly produced by Apis mellifera bees in the presence of different iron concentrations (ferrous sulfate heptahydrate-0, 25, 50, and 100 mg L-1). Two-dimensional electrophoresis was used for the fractionation of royal jelly proteins, and iron level was quantified using flame atomic absorption spectrometry technique. The proteins were identified using electrospray ionisation mass spectrometry. Analysis of variance followed by the Tukey test (P < 0.05) was utilised. Dietary supplementation with mineral Fe affected the protein content and number of proteins in the experimental period. Further, the diet containing the highest iron concentration showed a greater number of spots containing iron, as well as in the abdomen of the bees. The most protein containing Fe were classified as major royal jelly proteins. These results showed that Fe influenced the quality of royal jelly and can improve its nutritional value.
Assuntos
Ácidos Graxos/química , Compostos Ferrosos/análise , Proteínas de Insetos/análise , Animais , Abelhas , Suplementos Nutricionais , Ácidos Graxos/biossíntese , Compostos Ferrosos/administração & dosagem , Compostos Ferrosos/metabolismo , Proteínas de Insetos/metabolismoRESUMO
Because of strong impact of omics in many fields, and the complexity of the samples when focusing on areas such as genomics, (metallo)proteomics, metabolomics, among others, it is easy to rationalize the great importance that sample preparation has for achieving reliable results, mainly considering plant science. Then, this chapter points out applications of the sample preparation focusing on such areas, and a diversity of strategies, techniques, and procedures is highlighted and commented.
Assuntos
Genômica , Metabolômica , Plantas , Proteômica , Manejo de Espécimes/métodosRESUMO
Searching for prospective vanadium-based agents against Trypanosoma cruzi, the parasite causing Chagas disease, four new [VVO(8HQ-H)(L-2H)] compounds, where 8HQ is 8-hydroxyquinoline and L are tridentate salicylaldehyde semicarbazone derivatives L1-L4, were synthesized and characterized in the solid state and in solution. The compounds were evaluated on T. cruzi epimastigotes (CL Brener) as well as on VERO cells, as mammalian cell model. Compounds showed activity against T. cruzi (IC50 6.2-10.5 µM) of the same order than Nifurtimox and 8HQ, and a four- to sevenfold activity increase with respect to the free semicarbazones. For comparison, [VVO2(L-H)] series was prepared and the new [VVO2(L3-H)] was fully characterized. They showed negligible activity and low selectivity towards the parasite. The inclusion of 8HQ as ligand in [VVO(8HQ-H)(L-2H)] compounds led to good activities and increased selectivity towards the parasite with respect to 8HQ. 51V NMR experiments, performed to get insight into the nature of the active species, suggested partial decomposition of the compounds in solution to [VVO2(L-H)] and 8HQ. Depending on the dose, the compounds act as trypanocide or trypanostatic. A high uptake of vanadium in the parasites (58.51-88.9% depending on dose) and a preferential accumulation in the soluble protein fraction of the parasite was determined. Treated parasites do not seem to show a late apoptotic/necrotic phenotype suggesting a different cell death mechanism. In vivo toxicity study on zebrafish model showed no toxicity up to a 25 µM concentration of [VVO(8HQ-H)(L1-2H)]. These compounds could be considered prospective anti-T. cruzi agents that deserve further research.
Assuntos
Complexos de Coordenação/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Vanádio/química , Animais , Apoptose/efeitos dos fármacos , Chlorocebus aethiops , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Testes de Sensibilidade Parasitária , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/toxicidade , Células Vero , Peixe-ZebraRESUMO
Biochemical imbalances, provoked by aging or a secondary illness, might directly affect the brain, causing severe problems, such as loss of memory or alteration of behavior patterns. Brain disorders are usually classified as injuries (such as stroke, hematomas, and concussions), tumors, and neurodegenerative (such as Parkinson's and Alzheimer's diseases) and mental (such as depression, bipolar disorder, schizophrenia) diseases. As the pathophysiology of these illnesses is not completely established and multiple factors are involved, metallomics, a bioanalytical strategy that allows the detection of metal ions and metalloproteins in diverse biological matrices, is of extreme relevance in identifying which elements are affected by a disease and/or treatment. Thus, determining which element ions suffer disturbances in their homeostasis during the disease progress is relevant to understand the biochemical changes and propose new drug targets. In addition, it is well known that oxidative stress plays an important role in the development of pathological neurodegenerative and mental diseases, which may be caused by metal ion dyshomeostasis, so it is also important to understand endogenous antioxidant metalloprotein and metalloenzyme mechanisms in this regard. In this context, recent applications of metallomics in the study of neurodegenerative and mental disorders are discussed in this chapter, as well as future trends in this research area.
Assuntos
Antioxidantes/metabolismo , Homeostase , Transtornos Mentais/metabolismo , Metaloproteínas/metabolismo , Metais/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Humanos , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologiaRESUMO
This chapter focuses on bioimaging in metallomics, which involves metal and metalloids distribution in animal tissues. It starts with laser ablation-inductively coupled plasma-mass spectrometry followed by secondary ion mass spectrometry, synchrotron-based X-ray fluorescence, and electron microscopy, including transmission electron microscopy, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. The basic principles of these techniques and their application for qualitative and quantitative imaging of elements are presented. Sample preparation for bioimaging is briefly discussed. The usefulness of element bioimaging is demonstrated for cells and several animal tissues, including the brain, liver, hair, eye, teeth, and bone. As such, this chapter addresses the state of the art in bioimaging metallomics.
Assuntos
Diagnóstico por Imagem/métodos , Metaloides/metabolismo , Metais/metabolismo , Animais , HumanosRESUMO
Mercury is a potentially toxic element that is present in the environment of the Brazilian Amazon and is responsible for adverse health effects in humans. This study sought to assess possible protein biomarkers of mercury exposure in breast milk samples from lactating women in the Madeira and Negro Rivers in the Brazilian Amazon. The mercury content of hair samples of lactating women was determined, and the proteome of breast milk samples was obtained using two-dimensional electrophoresis after protein precipitation with acetone. Mercury measurements of protein spots obtained via protein fractionation were performed by graphite furnace atomic absorption spectrometry (GFAAS), and it was observed that mercury is linked to proteins with molecular masses in the range of 14-26 kDa. The total mercury concentration was also determined by GFAAS in unprocessed milk, lyophilized milk, and protein pellets, with the purpose of determining the mercury mass balance in relation to the concentration of this element in milk and pellets. Approximately 85 to 97% of mercury present in the lyophilized milk from samples of lactating women of the Madeira River is bound in the protein fraction. From lactating women of the Negro River, approximately 49% of the total mercury is bound in the protein fraction, and a difference of 51% is bound in the lipid fraction.
Assuntos
Cabelo/química , Mercúrio/análise , Metaloproteínas/análise , Leite Humano/química , Brasil , Feminino , HumanosRESUMO
Antipsychotics are the main line of treatment for schizophrenia, a disorder that affects about 1% of the worldwide population. Considering the poor performance of antipsychotics on patients, this work aimed at detecting alterations in the elemental profile resulting from the use of this type of medication using an elemental fingerprinting strategy. We evaluated 56 plasma samples from schizophrenia patients by inductively coupled plasma mass spectrometry (ICP-MS) before (t0) and after 6 weeks (t6) of treatment. The level of response of the patients (good vs. poor responders) and the medications taken were considered. Zinc, aluminum, phosphorus, and iron levels were found to be increased, whereas sodium, potassium, calcium, and magnesium levels decreased after treatment. Aluminum presented a higher level in poor responders at t0 when compared to good responders. At t6, iron showed an increased level when compared to t0 for good responders; however, its level remained constant in poor responders. The results of this exploratory study provide clues for further investigations on the role of metal ions in the treatment of schizophrenia.
Assuntos
Antipsicóticos/farmacologia , Análise Química do Sangue , Sangue/efeitos dos fármacos , Espectrometria de Massas/métodos , Esquizofrenia/sangue , Esquizofrenia/dietoterapia , Espectrofotometria Atômica/métodos , Adulto , Estudos de Coortes , HumanosRESUMO
This study presents data on the extraction and characterization of proteins associated with mercury in the muscle and liver tissues of jaraqui (Semaprochilodus spp.) from the Madeira River in the Brazilian Amazon. Protein fractionation was carried out by two-dimensional electrophoresis (2D-PAGE). Mercury determination in tissues, pellets, and protein spots was performed by graphite furnace atomic absorption spectrometry (GFAAS). Proteins in the spots that showed mercury were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The highest mercury concentrations were found in liver tissues and pellets (426 ± 6 and 277 ± 4 µg kg-1), followed by muscle tissues and pellets (132 ± 4 and 86 ± 1 µg kg-1, respectively). Mercury quantification in the protein spots allowed us to propose stoichiometric ratios in the range of 1-4 mercury atoms per molecule of protein in the protein spots. The proteins characterized in the analysis by ESI-MS/MS were keratin, type II cytoskeletal 8, parvalbumin beta, parvalbumin-2, ubiquitin-40S ribosomal S27a, 39S ribosomal protein L36 mitochondrial, hemoglobin subunit beta, and hemoglobin subunit beta-A/B. The results suggest that proteins such as ubiquitin-40S ribosomal protein S27a, which have specific domains, possibly zinc finger, can be used as biomarkers of mercury, whereas mercury and zinc present characteristics of soft acids.
Assuntos
Caraciformes/metabolismo , Proteínas de Peixes/metabolismo , Fígado/metabolismo , Mercúrio/toxicidade , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismoRESUMO
In the last decade, the development of diverse bioanalytical methodologies based on mass spectrometry imaging has increased, as has their application in biomedical questions. The distribution analysis of elements (metals, semimetals, and non-metals) in biological samples is a point of interest in life sciences, especially within the context of metallomics, which is the scientific field that encompasses the global analysis of the entirety of elemental species inside a cell or tissue. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been efficiently employed to generate qualitative and quantitative maps of elemental distribution in thin tissue sections of a variety of biological samples, for example, brain, cartilage, spinal cord, etc. The combination of elemental with molecular mass spectrometry allows obtaining information about the elements bound to proteins, when they are previously separated by gel electrophoresis (metalloproteomics), and also adding a new dimension to molecular mass spectrometry imaging by the correlation of molecular and elemental distribution maps in definite regions in a biological tissue. In the present review, recent biomedical applications in LA-ICP-MS imaging as a stand-alone technique and in combination with molecular mass spectrometry imaging techniques are discussed. Applications of LA-ICP-MS in the study of neurodegenerative diseases, distribution of contrast agents and metallodrugs, and metalloproteomics will be focused in this review. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:47-57, 2017.
Assuntos
Encefalopatias/diagnóstico por imagem , Meios de Contraste/análise , Espectrometria de Massas/métodos , Animais , Pesquisa Biomédica/métodos , Química Encefálica , Humanos , Terapia a Laser , Espectrometria de Massas/instrumentação , Metais/análise , Imagem Molecular/métodos , Proteômica/métodosRESUMO
This paper presents the results of mercury fractionation in muscle samples of dourada (Brachyplatystoma rousseauxii) from the JIRAU Hydroelectric Power Plant in the Madeira River Basin in the Amazon region of Brazil. The proteome of the dourada muscle was separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). The mercury present in the protein spots was determined by graphite furnace atomic absorption spectrometry (GFAAS) after acid mineralisation in an ultrasound bath. The protein spots in which the presence of mercury was detected were characterised by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) after tryptic digestion. The GFAAS determinations indicated that 65% of the mercury was linked to the protein fraction with a molar mass (Mm) of less than 90 kDa. The mercury concentrations in the seven spots in which this protein fraction was present were in the range of 11.40-35.10 µg kg(-1). Based on the mercury concentrations, it was possible to estimate that the protein spots contained approximately 1-3 mercury atoms per protein molecule. The ESI-MS/MS analysis allowed characterisation of the seven protein spots as the following proteins: protein NLRC5 (molar mass=18.10, pI=6.30); 39S ribosomal protein L36 mitochondrial (molar mass=15.40, pI=8.23); N-alpha-acetyltransferase 20 (Mm=15.95, pI=8.80); Mth938 domain-containing protein (Mm=15.01, pI=9.60); ubiquitin-40S ribosomal protein S27a (Mm=9.80, pI=7.60); parvalbumin alpha (Mm=12.40, pI=3.80) and parvalbumin beta (Mm=13.10, pI=3.45).
Assuntos
Proteínas de Peixes/isolamento & purificação , Mercúrio/isolamento & purificação , Músculos/química , Proteoma/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Animais , Brasil , Peixes-Gato/metabolismo , Eletroforese em Gel Bidimensional , Proteínas de Peixes/química , Contaminação de Alimentos/análise , Proteoma/química , Rios , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria AtômicaRESUMO
The main goal of this work is to evaluate some differential protein species in transgenic (T) and nontransgenic (NT) Arabidopsis thaliana plants after their cultivation in the presence or absence of sodium selenite. The transgenic line was obtained through insertion of CaMV 35S controlling nptII gene. Comparative proteomics through 2D-DIGE is carried out in four different groups (NT × T; NT × Se-NT (where Se is selenium); Se-NT × Se-T, and T × Se-T). Although no differential proteins are achieved in the T × Se-T group, for the others, 68 differential proteins (by applying a regulation factor ≥1.5) are achieved, and 27 of them accurately characterized by ESI-MS/MS. These proteins are classified into metabolism, energy, signal transduction, disease/defense categories, and some of them are involved in the glycolysis pathway-Photosystems I and II and ROS combat. Additionally, laser ablation imaging is used for evaluating the Se and sulfur distribution in leaves of different groups, corroborating some results obtained and related to proteins involved in the glycolysis pathway. From these results, it is possible to conclude that the genetic modification also confers to the plant resistance to oxidative stress.