RESUMO
BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG) has been repeatedly proposed as a potential plasma biomarker for myelodysplastic syndrome (MDS). OBJECTIVE: The goal of our work was to establish the total LRG plasma level and LRG posttranslational modifications (PTMs) as a suitable MDS biomarker. METHODS: The total plasma LRG concentration was determined with ELISA, whilst the LRG-specific PTMs and their locations, were established using mass spectrometry and public mass spectrometry data re-analysis. Homology modelling and sequence analysis were used to establish the potential impact of PTMs on LRG functions via their impact on the LRG structure. RESULTS: While the results showed that the total LRG plasma concentration is not a suitable MDS marker, alterations within two LRG sites correlated with MDS diagnosis (p= 0.0011). Sequence analysis and the homology model suggest the influence of PTMs within the two LRG sites on the function of this protein. CONCLUSIONS: We report the presence of LRG proteoforms that correlate with diagnosis in the plasma of MDS patients. The combination of mass spectrometry, re-analysis of publicly available data, and homology modelling, represents an approach that can be used for any protein to predict clinically relevant protein sites for biomarker research despite the character of the PTMs being unknown.
Assuntos
Glicoproteínas , Síndromes Mielodisplásicas , Biomarcadores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Leucina/metabolismo , Espectrometria de Massas , Síndromes Mielodisplásicas/diagnóstico , Processamento de Proteína Pós-TraducionalRESUMO
We describe the internal structure, spatial organization and dynamic formation of coronary artery thrombi from ST-segment elevation myocardial infarction patients. Scanning electron microscopy (SEM) revealed significant differences among four groups of patients (<2 hours; 2-6 hours; 6-12 hours, and >12 hours) related to the time of ischemia. Coronary artery thrombi from patients presenting less than 2 hours after the infarction were almost entirely composed of platelets, with small amounts of fibrin and red blood cells. In contrast, thrombi from late presenters (>12 hours) consisted of mainly platelets at the distal end, where clotting was initiated, with almost no platelets at the proximal end, while the red blood cell content went from low at the initiating end to more than 90% at the proximal end. Furthermore, fibrin was present mainly on the outside of the thrombi and older thrombi contained thicker fibers. The red blood cells in late thrombi were compressed to a close-packed, tessellated array of polyhedral structures, called polyhedrocytes. Moreover, there was redistribution from the originally homogeneous composition to fibrin and platelets to the outside, with polyhedrocytes on the interior. The presence of polyhedrocytes and the redistribution of components are signs of in vivo clot contraction (or retraction). These results suggest why later thrombi are resistant to fibrinolytic agents and other treatment modalities, since the close-packed polyhedrocytes form a nearly impermeable seal. Furthermore, it is of particular clinical significance that these findings suggest specific disparate therapies that will be most effective at different stages of thrombus development.
Assuntos
Plaquetas/patologia , Trombose Coronária , Eritrócitos/patologia , Fibrina/análise , Fibrinolíticos , Infarto do Miocárdio com Supradesnível do Segmento ST , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Trombose Coronária/diagnóstico por imagem , Trombose Coronária/tratamento farmacológico , Trombose Coronária/metabolismo , Trombose Coronária/patologia , Resistência a Medicamentos/fisiologia , Feminino , Fibrinolíticos/administração & dosagem , Fibrinolíticos/efeitos adversos , Humanos , Masculino , Microscopia Eletrônica de Varredura/métodos , Pessoa de Meia-Idade , Infarto do Miocárdio com Supradesnível do Segmento ST/etiologia , Infarto do Miocárdio com Supradesnível do Segmento ST/terapia , Trombectomia/métodos , Fatores de Tempo , Tempo para o TratamentoRESUMO
Fibrinogen is an abundant blood plasma protein that, inter alia, participates in blood coagulation. It polymerizes to form a fibrin clot that is among the major components of the thrombus. Fibrinogen reactions with various reactive metabolites may induce post-translational modifications (PTMs) into the protein structure that affect the architecture and properties of fibrin clots. We reviewed the previous literature to find the positions of PTMs of fibrinogen. For 7 out of 307 reported PTMs, we used molecular dynamics simulations to characterize their effect on the behavior of the fibrinogen coiled-coil domain. Interactions of the γ-coil with adjacent chains give rise to π-helices in Aα and Bß chains of even unmodified fibrinogen. The examined PTMs suppress fluctuations of the γ-coil, which may affect the fibrinolysis and stiffness of the fibrin fibers. Citrullination of AαR104 and oxidations of γP70 and γP76 to glutamic semialdehyde unfold the α-helical structure of Aα and Bß chains. Oxidation of γM78 to methionine sulfoxide induces the formation of an α-helix in the γ-coil region. Our findings suggest that certain PTMs alter the protein secondary structure. Thus, the altered protein structure may indicate the presence of PTMs in the molecule and consequently of certain metabolites within the system.
RESUMO
Atherosclerosis is a leading cause of major vascular events, myocardial infarction, and ischemic stroke. Tryptophan (TRP) catabolism was recognized as an important player in inflammation and immune response having together with oxidative stress (OS) significant effects on each phase of atherosclerosis. The aim of the study is to analyze the relationship of plasma levels of TRP metabolites, inflammation, and OS in patients with neurovascular diseases (acute ischemic stroke (AIS), significant carotid artery stenosis (SCAS)) and in healthy controls. Blood samples were collected from 43 patients (25 with SCAS, 18 with AIS) and from 25 healthy controls. The concentrations of twelve TRP metabolites, riboflavin, neopterin (NEO, marker of inflammation), and malondialdehyde (MDA, marker of OS) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Concentrations of seven TRP metabolites (TRP, kynurenine (KYN), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), anthranilic acid (AA), melatonin (MEL), tryptamine (TA)), NEO, and MDA were significantly different in the studied groups. Significantly lower concentrations of TRP, KYN, 3-HAA, MEL, TA, and higher MDA concentrations were found in AIS compared to SCAS patients. MDA concentration was higher in both AIS and SCAS group (p < 0.001, p = 0.004, respectively) compared to controls, NEO concentration was enhanced (p < 0.003) in AIS. MDA did not directly correlate with TRP metabolites in the study groups, except for 1) a negative correlation with kynurenine acid and 2) the activity of kynurenine aminotransferase in AIS patients (r = -0.552, p = 0.018; r = -0.504, p = 0.033, respectively). In summary, TRP metabolism is clearly more deregulated in AIS compared to SCAS patients; the effect of TRP metabolites on OS should be further elucidated.
RESUMO
Oxidative stress in humans is related to various pathophysiological processes, which can manifest in numerous diseases including cancer, cardiovascular diseases, and Alzheimer's disease. On the atomistic level, oxidative stress causes posttranslational modifications, thus inducing structural and functional changes into the proteins structure. This study focuses on fibrinogen, a blood plasma protein that is frequently targeted by reagents causing posttranslational modifications in proteins. Fibrinogen was in vitro modified by three reagents, namely sodium hypochlorite, malondialdehyde, and 3-morpholinosydnonimine that mimic the oxidative stress in diseases. Newly induced posttranslational modifications were detected via mass spectrometry. Electron microscopy was used to visualize changes in the fibrin networks, which highlight the extent of disturbances in fibrinogen behavior after exposure to reagents. We used molecular dynamics simulations to observe the impact of selected posttranslational modifications on the fibrinogen structure at the atomistic level. In total, 154 posttranslational modifications were identified, 84 of them were in fibrinogen treated with hypochlorite, 51 resulted from a reaction of fibrinogen with malondialdehyde, and 19 were caused by 3-morpholinosydnonimine. Our data reveal that the stronger reagents induce more posttranslational modifications in the fibrinogen structure than the weaker ones, and they extensively alter the architecture of the fibrin network. Molecular dynamics simulations revealed that the effect of posttranslational modifications on fibrinogen secondary structure varies from negligible alternations to serious disruptions. Among the serious disruptions is the oxidation of γR375 resulting in the release of Ca2+ ion that is necessary for appropriate fibrin fiber formation. Folding of amino acids γE72-γN77 into a short α-helix is a result of oxidation of γP76 to glutamic acid. The study describes behaviour of fibrinogen coiled-coil connecter in the vicinity of plasmin and hementin cleavage sites.
Assuntos
Fibrinogênio/química , Fibrinogênio/metabolismo , Processamento de Proteína Pós-Traducional , Humanos , Simulação de Dinâmica Molecular , Estrutura Secundária de ProteínaRESUMO
The onset and progression of numerous serious diseases (e.g., various types of malignancies, neurodegenerative diseases, and cardiac diseases) are, on a molecular level, associated with protein modifications and misfolding. Current methods for the detection of misfolded proteins are not able to detect the whole misfolded subproteome and, moreover, are rather laborious and time consuming. Herein, we report on a novel simple method for the detection of misfolded proteins employing a surface plasmon resonance (SPR) biosensor and heat shock protein 70 (Hsp70) that recognizes and traps misfolded proteins in a nucleotide-dependent manner. We use this method for the detection of misfolded proteins in blood plasma of patients with various subtypes of myelodysplastic syndromes (MDS) and healthy donors. Our results reveal significantly elevated levels of misfolded proteins in the two stages of MDS that are most affected by oxidative stress: low-risk (RARS) and intermediate-risk (RCMD) patients. This approach can be extended to a variety of diseases and provides unique insights into the thus far unexplored area of blood proteome.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Síndromes Mielodisplásicas/metabolismo , Dobramento de Proteína , Ressonância de Plasmônio de Superfície/métodos , Proteínas Sanguíneas/química , Proteínas de Choque Térmico HSP70/química , Humanos , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/diagnóstico , Estresse OxidativoRESUMO
Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies with a high risk of transformation to acute myeloid leukemia (AML). MDS are associated with posttranslational modifications of proteins and variations in the protein expression levels. In this work, we present a novel interactomic diagnostic method based on both protein array and surface plasmon resonance biosensor technology, which enables monitoring of protein-protein interactions in a label-free manner. In contrast to conventional methods based on the detection of individual biomarkers, our presented method relies on measuring interactions between arrays of selected proteins and patient plasma. We apply this method to plasma samples obtained from MDS and AML patients, as well as healthy donors, and demonstrate that even a small protein array comprising six selected proteins allows the method to discriminate among different MDS subtypes and healthy donors.
Assuntos
Síndromes Mielodisplásicas/diagnóstico , Mapeamento de Interação de Proteínas , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Análise de Componente Principal , Ligação Proteica , Ressonância de Plasmônio de Superfície , Adulto JovemRESUMO
BACKGROUND: Azacitidine (AZA) is a nucleoside analog used for treatment of myelodysplasia and the prediction of AZA responsiveness is important for the therapy management. METHODS: Using microarrays and reverse-transcription quantitative-PCR, we analyzed microRNA (miRNA) expression in bone marrow CD34+ cells of 27 patients with higher-risk myelodysplastic syndromes or acute myeloid leukemia with myelodysplasia-related changes before and during AZA treatment. RESULTS: At baseline, we found that future overall response rate was significantly higher in patients with upregulated miR-17-3p and downregulated miR-100-5p and miR-133b. Importantly, the high level of miR-100-5p at baseline was associated with shorter overall survival (HR = 4.066, P= 0.008). After AZA treatment, we observed deregulation of 30 miRNAs in responders (including downregulation of miR-10b-5p, miR-15a-5p/b-5p, miR-24-3p, and miR-148b-3p), while their levels remained unchanged in non-responders. CONCLUSIONS: Our study demonstrates that responders and non-responders have distinct miRNA patterns and that the level of specific miRNAs before therapy may predict the efficacy of AZA treatment.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , MicroRNAs/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Idoso , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologiaRESUMO
BACKGOUND: It has been indicated in plasma proteomic studies on different myelodysplastic syndrome (MDS) cohorts that alpha-2-HS-glycoprotein could be a promising MDS biomarker candidate. OBJECTIVE: The goal of this work was to estimate alpha-2-HS-glycoprotein (AHSG) plasma levels and its biomarker value in the low- and high-risk subgroups of MDS patients. METHODS: The level of AHSG was estimated for 115 plasma samples using ELISA. RESULTS: The AHSG plasma level was found to be decreased significantly (p= 2.59 × 10-7) in MDS patients (515 ± 58 µg/ml) when compared to healthy controls (579 ± 64 µg/ml). Pearson and Spearman correlation analyses showed that age is the principal factor affecting the AHSG plasma level, rather than risk/diagnosis in MDS. CONCLUSIONS: In this work we demonstrate that although the total plasma level of AHSG is decreased in myelodysplastic syndrome patients, in particular in advanced MDS, that decrease correlates more strongly with age than with diagnosis within our studied cohort. Thus, according to the AHSG data gathered so far, AHSG total plasma level does not seem to be a suitable MDS biomarker, but its particular proteoforms should be considered for the next steps in MDS research.
Assuntos
Biomarcadores/sangue , Síndromes Mielodisplásicas/sangue , alfa-2-Glicoproteína-HS , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Adulto JovemAssuntos
Proteínas Sanguíneas/metabolismo , Eritrócitos/metabolismo , Síndromes Mielodisplásicas/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/tratamento farmacológico , Processamento de Proteína Pós-TraducionalRESUMO
In recent years the plasma proteomes of several different myelodysplastic syndrome (MDS) subgroups have been investigated and compared with those of healthy donors. However, the resulting data do not facilitate a direct and statistical comparison of the changes among the different MDS subgroups that would be useful for the selection and proposal of diagnostic biomarker candidates. The aim of this work was to identify plasma protein biomarker candidates for different MDS subgroups by reanalyzing the proteomic data of four MDS subgroups: refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia or refractory anemia with ringed sideroblasts (RA-RARS), refractory anemia with excess blasts subtype 1 (RAEB-1), and refractory anemia with excess blasts subtype 2 (RAEB-2). Reanalysis of a total of 47 MDS patients revealed biomarker candidates, with alpha-2-HS-glycoprotein and leucine-rich alpha-2-glycoprotein as the most promising candidates.
Assuntos
Glicoproteínas/sangue , Síndromes Mielodisplásicas/sangue , alfa-2-Glicoproteína-HS/metabolismo , Adulto , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Our aim was to search for proteome changes in peripheral blood mononuclear cells (PBMCs) of MDS patients with refractory cytopenia with multilineage dysplasia. PBMCs were isolated from a total of 12 blood samples using a Histopaque-1077 solution. The proteins were fractioned, separated by 2D SDS-PAGE (pI 4-7), and double-stained. The proteomes were compared and statistically processed with Progenesis SameSpots; then proteins were identified by nano-LC-MS/MS. Protein functional association and expression profiles were analyzed using the EnrichNet application and Progenesis SameSpots hierarchical clustering software, respectively. By comparing the cytosolic, membrane, and nuclear fractions of the two groups, 178 significantly (P < 0.05, ANOVA) differing spots were found, corresponding to 139 unique proteins. Data mining of the Reactome and KEGG databases using EnrichNet highlighted the possible involvement of the identified protein alterations in apoptosis, proteasome protein degradation, heat shock protein action, and signal transduction. Western blot analysis revealed underexpression of vinculin and advanced fragmentation of fermitin-3 in MDS patients. To the best of our knowledge, this is the first time that proteome changes have been identified in the mononuclear cells of MDS patients. Vinculin and fermitin-3, the proteins involved in cell adhesion and integrin signaling, have been shown to be dysregulated in MDS.
Assuntos
Bases de Dados de Proteínas , Leucócitos Mononucleares/metabolismo , Síndromes Mielodisplásicas/metabolismo , Proteoma/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.
Assuntos
Acrilamidas/química , Técnicas Biossensoriais/instrumentação , MicroRNAs/análise , MicroRNAs/química , Polímeros/química , Ressonância de Plasmônio de Superfície/instrumentação , Fracionamento Celular , Materiais Revestidos Biocompatíveis/síntese química , Misturas Complexas/análise , Desenho de Equipamento , Análise de Falha de Equipamento , MicroRNAs/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Fast-staining protocols based on the use of Coomassie blue dye for SDS-PAGE separated proteins, represent a quick and simple solution for protein visualization. It has been shown however, that in some cases a phenomenon of missing spots or spot discoloration may be observed in the proteome pattern when the standard fast-staining protocol is used. In this work, it is demonstrated that this occurrence is affected by the biological variability of samples, and therefore, cannot be observed in all samples. Moreover, it is demonstrated that the phenomenon is manifested exclusively in nonfixed gels, and that including a fixation step into the fast-staining protocol prevented this phenomenon. In conclusion, it has been demonstrated that standard Coomassie blue dye based fast staining for SDS-PAGE resolved proteins is affected by the biological variability of samples in nonfixed gels.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Corantes de Rosanilina/química , Eletroforese em Gel Bidimensional , Imidazóis , Proteínas/análise , Proteínas/química , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: Congenital dysfibrinogenemia and hypofibrinogenemia are rare diseases characterized by inherited abnormality in the fibrinogen molecule, resulting in functional defects (dysfibrinogenemia) or low fibrinogen plasma levels (hypofibrinogenemia). MATERIALS AND METHODS: We have described two abnormal fibrinogens - fibrinogen Hranice (γ Phe204Val) and Praha IV (γ Ser313Gly). The carrier of the Hranice mutation was a 40-year-old female with low fibrinogen levels. The carrier of the Praha IV mutation was a 42-year-old man with a history of idiopathic thrombosis, low functional fibrinogen levels, and a prolonged thrombin time. RESULTS: Fibrin polymerization kinetics measurement was normal in both cases (after the addition of either thrombin or reptilase), as well as was fibrinolysis. Scanning electron microscopy and confocal microscopy revealed significantly wider fibers in both cases, when compared with fibers prepared from healthy control samples. Although both cases are situated in the γ-nodule, they manifested differently. While the γ Ser313Gly mutation manifested as dysfibrinogenemia with a thrombotic background, the γ Phe204Val mutation manifested as hypofibrinogenemia without clinical symptoms. The mutation sites of both fibrinogens are in highly conserved regions of the fibrinogen γ chains. γ Ser313 is situated in a class 16:18 ß hairpin and is involved in hydrogen bonding with γ Asp320. γ Phe204 is situated in an inverse γ turn and may be involved in π-π interactions. CONCLUSIONS: Both mutations cause conformational changes in fibrinogen, which lead either to impaired fibrinogen assembly (fibrinogen Hranice) or abnormal fibrinogen function (fibrinogen Praha IV).
Assuntos
Afibrinogenemia/congênito , Fibrinogênio/genética , Fibrinogênios Anormais/genética , Mutação Puntual , Adulto , Afibrinogenemia/sangue , Afibrinogenemia/genética , Afibrinogenemia/metabolismo , Feminino , Fibrina/genética , Fibrina/metabolismo , Fibrina/ultraestrutura , Fibrinogênio/metabolismo , Fibrinogênio/ultraestrutura , Fibrinogênios Anormais/metabolismo , Fibrinogênios Anormais/ultraestrutura , Fibrinólise , Humanos , MasculinoRESUMO
The goal of this study was to explore the plasma proteome of myelodysplastic syndrome (MDS) patients with refractory anemia with excess blasts subtype 2 (RAEB-2) in comparison to healthy controls. 20 plasma samples were separated with 2D electrophoresis and statistically processed with Progenesis SameSpots software. 47 significantly differing (P < 0.05) spots were observed, and 27 different proteins were identified by nano-LC-MS/MS. Mass spectrometry-based relative label-free quantification showed a 2-fold increase of the leucine-rich alpha-2-glycoprotein (LRAG) peptide levels in the RAEB-2 group. Changes in the fragments of the inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) protein were observed. Western blot analysis showed no differences in albumin and ITIH4 levels, while increased expression was observed for LRAG in the RAEB-2 group. Quantification using ELISA showed decreased plasma level of alpha-2-HS glycoprotein in the RAEB-2 group. In conclusion, this is the first time that alpha-2-HS glycoprotein and LRAG were proposed as new biomarkers of RAEB-2 and advanced MDS, respectively. Alpha-2-HS glycoprotein, a protein involved in the bone marrow development and previously proposed as a MDS biomarker candidate, was significantly decreased in RAEB-2. Increased expression and changes in modification(s) were observed for LRAG, a protein involved in granulocytic and neutrophil differentiation, and angiogenesis.
Assuntos
Anemia Refratária com Excesso de Blastos/sangue , Proteínas Sanguíneas/metabolismo , Proteoma/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Biomarcadores/sangue , Biomarcadores/química , Proteínas Sanguíneas/química , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Síndromes Mielodisplásicas/sangue , Fragmentos de Peptídeos/química , Proteoma/química , Espectrometria de Massas em Tandem , Adulto JovemAssuntos
Afibrinogenemia/congênito , Fibrinogênios Anormais/genética , Mutação de Sentido Incorreto , Mutação Puntual , Adulto , Afibrinogenemia/genética , Doenças Assintomáticas , Testes de Coagulação Sanguínea , Dimerização , Éxons/genética , Feminino , Fibrinogênios Anormais/química , Heterozigoto , Humanos , Ligação de Hidrogênio , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Modelos Moleculares , Nefelometria e Turbidimetria , Conformação Proteica , Trombina/farmacologiaRESUMO
The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.