Proteomic and ionomic study for identification of biomarkers in biological fluid samples of patients with psychiatric disorders and healthy individuals : from sample preparation to quantification

Orientador: Marco Aurélio Zezzi ArrudaTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: O transtorno afetivo bipolar (TAB) é uma doença psiquiátrica caracterizada por alterações de humor que afeta milhares de pessoas no mundo. A escassez de conhecimentos científicos acerca de sua patogênese faz do TAB uma doença de complexa e de difícil diagnóstico. Alguns pacientes portadores do TAB são, por exemplo, diagnosticados equivocadamente como depressivos unipolares e, como consequência, são tratados ineficazmente. Diante desse fato, uma análise exploratória utilizando estratégias ômicas (proteômica e ionômica) comparativas no soro sanguíneo de pacientes com TAB, controles saudáveis, incluindo não familiares (CSNF) e familiares (CSF), pacientes com esquizofrenia (ESQ) e pacientes com outros transtornos mentais (OTM) foi realizado, a fim de determinar potenciais marcadores biológicos do TAB. Para o estudo proteômico, três métodos foram avaliados para simplificar o proteoma do soro sanguíneo: depleção de proteínas abundantes usando agentes químicos (acetonitrila, ACN; e ditiotreitol, DTT) em sequência; depleção de proteínas abundantes, usando nanopartículas magnéticas (MNPs) e a equalização de proteínas usando kit comercial ProteoMiner® (PM). Para o estudo ionômico, dois métodos foram testados para a decomposição da amostra: decomposição assistida por microondas (método convencional) e decomposição rápida assistida por ultrassom de alta potência (método proposto). Como resultado do estudo proteômico, o método PM apresentou-se como a melhor estratégia para remover as proteínas de alta abundância. Por meio da comparação de imagens de géis 2-D DIGE, 37 spots de proteínas foram encontrados como diferencialmente abundantes (p <0,05, teste t de Student), que exibiram variação do fator de regulação ? 2 vezes o valor médio das intensidades dos spots do soro de pacientes ESQ, TAB e OTM em comparação aos controles (CSF e CSNF). A partir desses spots detectados, foram identificadas 13 diferentes proteínas: ApoA1, ApoE, ApoC3, ApoA4, Samp, SerpinA1, TTR, IgK, Alb, VTN, TR, C4A e C4B. Como resultado do estudo ionômico, o método proposto usando ultrassom apresentou as melhores condições para a extração de Zn, Cu, Fe, Li, Cd e Pb do soro sanguíneo. A partir da aplicação do método proposto, as recuperações dos analitos variaram entre 80 a 121%, com desvio padrão relativo variando entre 3-10% (n=3). Assim, a aplicação das condições ótimas (40%(v/v) de HNO3 + 30%(v/v) de HCl como componentes da solução extratora, 60% como amplitude de sonicação e 3 min como tempo ótimo de sonicação) foram aplicadas nas amostras de soro de TAB, ESQ, e indivíduos saudáveis, onde observou-se a ausência de Pb e Cd para todas as amostras avaliadas, e diferenças significativas na concentração de Zn, Cu, Li e Fe entre os grupos avaliados. Para o grupo dos indivíduos com o diagnóstico de TAB, um alto nível da concentração dos metais foi observado, enquanto que para o grupo de ESQ, todos os metais foram encontrados em baixos níveis, quando comparados com os indivíduos saudáveis. Além disso, uma nova metodologia foi desenvolvida baseada em membrana polimérica a fim de extrair a totalidade das proteínas presentes em amostras de urina livres de interferentes, permitindo caracterizar o proteoma urinário com o objetivo de descobrir biomarcadores de doenças humanas. Nesse estudo, após otimização de alguns parâmetros, tais como composição da membrana polimérica (celulose, acetato de celulose, nitrocelulose), tamanho do poro da membrana (0,22 e 0,45 µm), vazão da urina (0,25, 0,5 e 1 mL -1) e pH do meio (3, 4, 5, 6, e 7), foi observado melhores resultados usando membrana de nitrocelulose, com um tamanho de poro de 0,22 µm, e vazão de urina de 0,25 mL min-1, além de usar um pH 3 para obter uma retenção de proteína urinária na membrana superior a 90%. Além disso, a metodologia desenvolvida foi aplicada a um estudo de classificação de gênero, a fim de avaliar uma diferenciação proteômica de gênero (masculino e feminino). Como resultado, uma classificação significativa foi obtida, de acordo com os dados obtidos por análise de componente principal (PCA) e hierarquical clastering, bem como vulcano plot e os gráficos de intersecçãoAbstract: Bipolar disorder (BD) is a psychiatric disorder characterized by mood alteration that affects thousands of people around the world. The little scientific knowledge about its pathogenesis, become BD a complex disease, and with hard diagnosis. Some patients with BD are, for example, misdiagnosed as unipolar depressives and, as a consequence, they are undergoing ineffective treatments. Thus, an exploratory analysis using strategies omics (proteomic and ionomic) in the blood serum of patients with BD, healthy controls, including nonfamily (HCNF) and familial (HCF), patients with schizophrenia (SCH) and patients with other mental disorders (OTM) were compared in order to determine potential biological markers of BD. For the proteomic study three methods were evaluated to simplify the blood serum proteome: sequential depletion of abundant proteins using chemical agents (acetonitrile, ACN, and dithiothreitol, DTT); abundant protein depletion using magnetic nanoparticles (MNPs) and the equalization of proteins using commercial ProteoMiner (PM) kit. For the ionomic study, two methods were tested for the decomposition of the sample: microwave assisted decomposition (conventional method); and fast decomposition assisted by high power ultrasound (proposed method). As a result of the proteomic study, the PM method was the best strategy to remove high abundance proteins. By comparing 2D-DIGE gel images, 37 protein spots were found to be differentially abundant (p <0.05, Student's t-test), which exhibited a variation in the regulation factor ? 2 times the mean value of the intensities of serum spots of ESQ, TAB and OTM patients compared to controls (HCF and HCNF). From these detected spots, 13 different proteins were identified: ApoA1, ApoE, ApoC3, ApoA4, Samp, SerpinA1, TTR, IgK, Alb, VTN, TR, C4A and C4B. As a result of the ionomic study, the method proposed using ultrasound presented the best conditions for the extraction of Zn, Cu, Fe, Li, Cd and Pb from serum. From the application of the proposed method, the recoveries of the analytes varied between 80 to 121%, with relative standard deviation varying between 3-10%. Thus, the application of the optimal conditions (40% (v/v) of HNO3 + 30% (v/v) HCl as components of the extractive solution, 60% as the sonication amplitude and 3 min as the optimal sonication time) in the serum samples from TAB, ESQ, and healthy individuals, where the absence of Pb and Cd was observed for all the samples evaluated and significant differences in the concentration of Zn, Cu, Li and Fe among the groups evaluated. To TAB group, the metals were observed at a high level, while for the ESQ group, all metals were found at low levels when compared to healthy individuals. In addition, as a result of the study during the sandwich period, a new methodology was developed based on polymer membrane in order to extract all the proteins present in urine samples interferents-free, allowing to characterize the proteome of urine with the objective of discovering biomarkers of human diseases. In this study, after optimization of some parameters, such as polymer membrane composition (cellulose, cellulose acetate, nitrocellulose), membrane pore size (0.22 and 0.45 ?m), urine flow rate (0.25, 5 and 1 mL -1) and pH of the medium (3, 4, 5, 6, and 7), it was observed that the best results were obtained using nitrocellulose membrane, with a pore size of 0.22 ?m and urine of 0.25 mL min-1, in addition to using a pH of 3 to obtain a retention of membrane urinary protein greater than 90%. In addition, the methodology developed was applied in a gender classification study, in order to evaluate a gender-based proteomic differentiation (male and female). As a result, a significant classification was obtained, according to data obtained by principal component analysis (PCA) and hierarchical clastering, as well as vulcano plot and intersection plotsDoutoradoQuimica AnaliticaDoutor em CiênciasCAPE

Avaliação de material alternativo para uso como adsorvente na extração em fase sólida de resíduos de pesticidas em estipe de coqueiro (Cocos nucifera L)

The coconut (Cocos nucifera L) is a rich source of nutrient and as well as other plants is under attack from different types of pests and diseases, causing losses to producers. Pesticide use remains the primary control strategy and combat pests and diseases in agricultural crops. However, the excessive and indiscriminate use of these products can cause serious harm to human health and the environment due to the high toxicity these products. In Brazil, until now no stipulated values of maximum residue limits (MRLs) for pesticides in coconut crops. Similarly, there is little information about the degree of contamination. Therefore analytical methodologies must be developed for the determination of pesticide residues in coconut cultivation, aiming to ensure human health. Thus, this work proposes a methodology to determine residue of bromopropylate, clofentezine, coumaphos, difenoxuron, diniconazole, fenvalerate, flumetralin, flumeturon, teflubenzuron in greens stipe, appling the matrix solid phase dispersion (MSPD) and liquid chromatography with diode array detector (LC-DAD) for analysis. In this study we evaluated the best sorbent/solvent combinations (using silica, Florisil, neutral alumina, C18 and coordination polymers [Zn(BDC)(H2O)2]n, [Zn(BDC)0.99(NH2-BDC)0.01)(H2O)2]n, [Zn(BDC)0.95(NH2-BDC)0.05)(H2O)2]n, [Zn(BDC)0.90(NH2-BDC)0.10)(H2O)2]n/acetonitrile), the best ratio of sample/ adsorbent mass, as volume of the elution solvent used for extraction were evaluated. Among the investigated combinations, the system comprising 0.25 g of stem, 1 g of neutral alumina and 5 mL of acetonitrile was the most efficient in recovery resulting from 70 and 93%, with relative standard between 1.6% and 10%, in the levels of concentration 0.04, 0.08, 0.2, 0.4, 2.0, 3.0; 4.0 ìg g-1. The linearity was above 0.9991 and the limits of detection between 0.01 and 0.05 ìg g-1 and quantification 0.05 and 0.2 ìg g-1. Among the polymeric phases, the [Zn(NH2BDC)0.1(BDC)0.9(H2O)2]n was most eficiente in the extraction of bromopropylate, clofentezine, coumaphos, difenoxuron, diniconazole, fenvalerate, flumetralin, flumeturon and teflubenzuron.O coqueiro (Cocos nucifera L.) e uma rica fonte de nutrientes e, assim como outras plantas e alvo de ataque de diferentes tipos de pragas e doencas, causando prejuizos aos produtores. O uso de pesticidas continua sendo a principal estrategia de controle e combate de pragas e doencas nas plantacoes agricolas. No entanto, o uso excessivo e indiscriminado desses produtos podem ocasionar serios danos a saude humana e ao ambiente, devido a elevada toxicidade desses produtos. No Brasil, ate o momento, nao ha valores estipulados de limites maximos de residuos (LMR) para pesticidas na cocoicultura. Do mesmo modo, ha pouca informacao quanto ao grau de contaminacao. Portanto metodologias analiticas tem de ser desenvolvidas para determinacao de residuos de pesticidas na cultura de coqueiro, com intuito de assegura essencialmente a saude humana. Nesse sentido, esse trabalho propoe uma metodologia que determine residuo de bromopropilato, clofentezina, cumafos, difenoxurom, diniconazol, fenvalerato, flumetralina, flumeturom, teflubenzurom em estipe de coqueiro, fazendo uso da tecnica de DMFS para extracao e CL-DAD para analise. Nesse estudo foram avaliados as melhores combinacoes sorvente/solvente (utilizando silica gel, Florisil, alumina neutra, C18, e polimeros de coordenacao [Zn(BDC)(H2O)2]n, [Zn(BDC)0,99(NH2-BDC)0,01)(H2O)2]n, [Zn(BDC)0,95(NH2-BDC)0,05)(H2O)2]n, [Zn(BDC)0,90(NH2-BDC)0,10)(H2O)2]n/acetonitrila). Avaliou-se a melhor relacao de massa amostra/adsorvente, alem do volume do solvente de eluicao utilizado para extracao. Dentre as combinacoes avaliadas, o sistema constituido de 0,25 g de estipe, 1 g de alumina neutra e 5 mL de acetonitrila foi o mais eficiente resultando em recuperacao entre 70 e 93% com precisao entre 1,6% e 10% nos niveis de concentracao 0,04, 0,08, 0,2, 0,4, 2,0, 3,0; 4,0 Êg g-1. As linearidades foram acima de 0,9991 e os limites de deteccao foram entre 0,01 e 0,05 Êg g-1 e de quantificacao entre 0,05 e 0,2 Êg g-1. Entre as fases polimericas estudadas, o [Zn(NH2BDC)0,1(BDC)0,9(H2O)2]n foi o mais eficiente na recuperacao de bromopropilato, clofentezina, cumafos, difenoxurom, diniconazol, fenvalerato, flumetralina, flumeturom e teflubenzurom

Quantifying proteins at microgram levels integrating gel electrophoresis and smartphone technology

A combination between gel electrophoresis and smartphone technology is applied for quantifying proteins in a serum sample. Electrophoresis not only allows one to separate different proteins but also to build-up a calibration curve for those proteins. To expand its applicability, a smartphone allows one to capture a gel image, through its camera. Additionally, the treatment of the data extracted through each protein band is performed using a freely available program (PhotoMetrix), which is downloaded to one's own smartphone. Through this approach, the quantification of proteins such as albumin, immunoglobulin, and carbonic anhydrase is performed in a serum sample by acquiring images using 32 × 32 pixels for each image in the region of the protein bands. An LOQ from 1.9 to 2.4 μg and r2 > 0.980 may be obtained. Comparing results through the analyses of the gels using Image Master 2D Platinum 6.0 software reflects good agreement (95% confidence level) between the results1984549CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP401170/2016-088887.115406/20152016/07384-7; 2018/00768-

Speciomics as a concept involving chemical speciation and omics

The study of chemical speciation and the refinement and expansion of omics-based methods are both consolidated and highly active research fields. Although well established, such fields are extremely dynamic and are driven by the emergence of new strategies and improvements in instrumentation. In the case of omics-based studies, subareas including lipidomics, proteomics, metallomics, metabolomics and foodomics have emerged. Here, speciomics is being proposed as an "umbrella" term, that incorporates all of these subareas, to capture studies where the evaluation of chemical species is carried out using omics approaches. This paper contextualizes both speciomics and the speciome, and reviews omics applications used for species identification through examination of proteins, metalloproteins, metabolites, and nucleic acids. In addition, some implications from such studies and a perspective for future development of this area are provided. SIGNIFICANCE: The synergic effect between chemical speciation and omics is highlighted in this work, demonstrating an emerging area of research with a multitude of possibilities in terms of applications and further developments. This work not only defines and contextualizes speciomics and individual speciomes, but also demonstrates with some examples the great potential of this new interdisciplinary area of research. [Abstract copyright: Copyright © 2022 Elsevier B.V. All rights reserved.

Multivariate optimization, in conjugation with matrix solid-phase dispersion and digital image detection, allows residual dye determination from shrimp samples

Here, we report an optimization of the extraction of crystal violet and congo red dyes from shrimp samples, employing matrix solid phase dispersion and improved multivariate optimization. Three factors were evaluated: (i) adsorbent nature (neutral alumina, silica gel and C18); (ii) eluent strength (dimethylformamide, methanol and acetonitrile); and (iii) sample-adsorbent ratio (1:1; 1:5; and 1:10, m/m). Successful extraction of dyes was achieved using dimethylformamide as eluent, neutral alumina as adsorbent and sample:adsorbent ratio of 1:5 (m/m). Adsorption and kinetic studies suggest strong interaction between dye and adsorbent. Furthermore, employing digital image colorimetric detection, the study observed limit of detection (LOD) ranging between 3.8 to 29.8 µg L − 1 and limit of quantification (LOQ) ranging between 12.3 to 90.3 µg L − 1 with R2 > 0.9927 for both dyes, CV and CR, respectively. Notably, recovery was greater than 86 % with a relative standard deviation (RSD) inferior to 7 % (n = 3). These findings indicate the effectiveness of the improved multivariate optimization technique in conjunction with matrix solid-phase dispersion for successful extraction and quantification of dyes from shrimp samples, showing its potential for accurate and sensitive detection in similar analyses. The proposed method was compared with the gold method (UV/Vis spectroscopy) and the statistical treatment showed that the results of both methods are comparable

Bipolar Disorder: Recent Advances And Future Trends In Bioanalytical Developments For Biomarker Discovery.

In this manuscript we briefly describe bipolar disorder (a depressive and manic mental disease), its classification, its effects on the patient, which sometimes include suicidal tendencies, and the drugs used for treatment. We also address the status quo with regard to diagnosis of bipolar disorder and recent advances in bioanalytical approaches for biomarker discovery. These approaches focus on blood samples (serum and plasma) and proteins as the main biomarker targets, and use various strategies for protein depletion. Strategies include use of commercially available kits or other homemade strategies and use of classical proteomics methods for protein identification based on bottom-up or top-down approaches, which used SELDI, ESI, or MALDI as sources for mass spectrometry, and up-to-date mass analyzers, for example Orbitrap. We also discuss some future objectives for treatment of this disorder and possible directions for the correct diagnosis of this still-unclear mental illness.407661-

Simplifying the human serum proteome for discriminating patients with bipolar disorder of other psychiatry conditions

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORAn exploratory analysis using proteomic strategies in blood serum of patients with bipolar disorder (BD), and with other psychiatric conditions such as Schizophrenia (SCZ), can provide a better understanding of this disorder, as well as their discriminati501811181125FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR2016/07384-7401170/2016-088887.115406/2015The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil, 2016/07384-7), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil, 401170/2016-0) and the Coordenação de Aperfei