Name: Isolamento, caracterização e análise proteômica de vesículas extracelulares derivadas de plasma para descoberta de biomarcadores cardiovasculares (Vídeo) | JoVE Journal
Uploaded: 2025-01-31T12:30:00.000+00:00
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Description: Aprenda a isolar e caracterizar vesículas extracelulares derivadas de plasma humano para descoberta de biomarcadores cardiovasculares. Este protocolo utiliza análise proteômica por LC-MS/MS para identificar proteínas relevantes em doenças cardíacas.

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<meta charset="utf8"></head><body>Estudos de biomarcadores cardiovasculares usando proteoma de vesícula extracelular plasmática

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</ol>

<table><tbody><tr><td>4x Laemmli sample bufffer</td><td>Bio-rad</td><td>1610747</td><td></td></tr><tr><td>10x RIPA-Buffer</td><td>Abcam</td><td>ab156034</td><td></td></tr><tr><td>26.3 mL Frasco de policarbonato com conjunto de tampa para ultracentrifugação</td><td>Centrífuga de bancada Beckman Coulter</td><td>355654</td><td></td></tr><tr><td>5810R</td><td>Eppendorf</td><td>5811000015</td><td></td></tr><tr><td>Acetonitrila (ACN)</td><td>Biosolve</td><td>0001204101BS</td><td></td></tr><tr><td>Ditiotreitol (DTT)</td> <td>Sigma-Aldrich</td><td>43816-10ML</td><td></td></tr><tr><td>Solução salina tamponada com fosfato de Dulbecco (PBS)</td> <td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Ácido fórmico 99% ULC / MS 100</td><td>Biosolve</td><td>0006914143BS</td><td></td></tr><tr><td>Histopaque - 1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Iodoacetamida (IAA)</td> <td>Sigma-Aldrich</td><td>I6125-5G</td><td></td></tr><tr><td>Espectrômetro de Massa Orbitrap Q Exactive HF</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>MOPS SDS buffer de corrida</td><td>Thermo Fisher</td><td>B0001</td><td></td></tr><tr><td>NanoSight NS300</td><td>Malvern Panalytical&nbsp;</td><td>Software NS300</td><td></td></tr><tr><td>Nanosight NTA 3.2</td><td>Malvern Panalytical&nbsp;</td><td></td><td></td></tr><tr><td>NuPAGE 4&nbsp; bis 12&nbsp;%, géis de proteína Bis-Tris</td><td>Invitrogen</td><td>NP0323</td><td></td></tr><tr><td>Optima XPN-80 ultracentrífuga de chão</td><td>Beckman Coulter</td><td>521-4180</td><td></td></tr><tr><td>PageRuler Plus Prestained Protein Ladder</td><td>Thermo Fisher Scientific</td><td>26619</td><td></td></tr><tr><td>Protease/Fosfatase Inhibitor Cocktail (100X)</td><td>Cell Signaling Technology</td><td>5872S</td><td></td></tr><tr><td>Marcador de proteína</td><td>Thermo Fisher</td><td>26616</td><td></td></tr><tr><td>Proteome Discoverer 2.5</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Q Exactive Plus Espectrômetro de Massa Híbrido Quadrupole-Orbitrap</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Coomasssie de mancha rápida</td><td>Serva</td><td>35081.01</td><td></td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 1.9 &micro; m 1 g</td><td>Dr. Maisch</td><td>r119.aq.0001</td><td></td></tr><tr><td>SDS-PAGE gel comercial</td><td>Thermo Fisher</td><td>NW00100BOX</td><td></td></tr><tr><td>S-Monovette&nbsp; Citrat 9NC 0.106 mol/l 3,2%</td><td>Sarstedt</td><td>02.1067.001</td><td></td></tr><tr><td>Concentrador de velocidade a vácuo</td><td>Savant</td><td></td><td></td></tr><tr><td>Swiss-Prot (Uniprot) Homo sapiens (UP000005640, junho de 2020) banco de dados de proteínas</td><td>UniProt</td><td>https://www.uniprot.org/proteomes/UP000005640</td><td></td></tr><tr><td>Tampão de bicarbonato de trietilamônio (TEAB)</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Ácido trifluoroacético (TFA) para HPLC >99%, 100 mL</td><td>Sigma-Aldrich</td><td>302031-100ML</td><td></td></tr><tr><td>Tripsina MS-Grade</td><td>Thermo Fisher</td><td>90058</td><td></td></tr><tr><td>Tipo 50.2 Ti Rotor de ângulo fixo</td><td>Beckman Coulter</td><td>337901</td><td></td></tr><tr><td>UHPLC Dionex Ultimate 3000</td><td>Thermo Fisher Scientific</td><td>ULTIM3000RSLCNANO</td><td></td></tr><tr><td>Water</td><td>Biosolve</td><td>0023214102BS</td><td></td></tr></tbody></table>

isolamento, caracterização e análise proteômica de vesículas extracelulares derivadas de plasma para descoberta de biomarcadores cardiovasculares

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

As EV foram isoladas de amostras de plasma (n = 3) de pacientes sem doença cardiovascular evidente, bem como de pacientes com infarto do miocárdio sem supradesnivelamento do segmento ST (IAMSSST; n = 2), usando o protocolo de ultracentrifugação diferencial estabelecido. A separação adequada do EV plasmático foi confirmada por immunoblotting das proteínas enriquecidas com EV TSG-101, anexina 5 (Anx5) e CD9 em isolados EV em comparação com o plasma depletado de EV dos mesmos pacientes (Figura 1A</stron...

Isolamento, caracterização e análise proteômica de vesículas extracelulares derivadas de plasma para descoberta de biomarcadores cardiovasculares

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

isolation-characterization-proteomic-analysis-plasma-derived

Research

JoVE Journal

Medicine

Medicina

Vesículas extracelulares presentes no sangue têm sido sugeridas como novos biomarcadores para várias doenças. Aqui, apresentamos um protocolo para o isolamento de grandes microvesículas derivadas da membrana plasmática de amostras de sangue periférico e sua posterior análise por citometria de fluxo convencional e Western Blotting.

Isolamento e caracterização de microvesículas a partir de sangue periférico

Aqui, apresentamos um protocolo para purificar exosomes do plasma e soro com reduzida co purificação de proteínas do sangue não-exosomal. O protocolo otimizado inclui ultrafiltração, tratamento com proteases e cromatografia de exclusão. Reforçada purificação de exosomes análises benefícios a jusante, incluindo a quantificação mais precisa de vesículas e caracterização proteômica.

Digestão de proteínas, ultrafiltração e cromatografia de exclusão para otimizar o isolamento do Exosomes do Plasma de sangue humano e soro

O protocolo descreve um método reprodutível, projetado para uso com sobrenadantes de cultura celular para detectar epítopos superficiais em pequenas vesículas extracelulares (EV). Ele utiliza a imunoprecipitação de EV específica usando grânulos juntamente com anticorpos que reconhecem o antígeno de superfície CD9, CD63 e CD81. O método é otimizado para análise de citometria de fluxo a jusante.

Fluxo Cytometric análise das vesículas extracelulares de célula-condicionado Media

O presente relatório destaca os requisitos cronológicos para o isolamento da vesícula extracelular (EV) de microglia ou macrófagos sanguíneos. Os EVs derivados de microglia foram avaliados como reguladores do crescimento do neurite, enquanto os EVs derivados do macrófago sanguíneo foram estudados no controle da invasão celular C6 glioma em ensaios in vitro. O objetivo é entender melhor essas funções de EV como mediadores imunológicos em microambientes específicos.

Caracterização de vesículas extracelulares derivadas de células imunes e estudo do impacto funcional no ambiente celular

O presente protocolo descreve um método para extração de vesículas extracelulares do sangue periférico e tecidos sólidos com posterior perfilamento de antígenos de superfície e cargas proteicas.

Isolamento e Análise de Vesículas Extracelulares Rastreáveis e Funcionalizadas do Plasma e Tecidos Sólidos

A última geração de ferramentas de caracterização de EV é capaz de uma única análise de EV em vários parâmetros simultaneamente. A citometria de nanofluxo mede todas as partículas biológicas maiores que 45 nm sem marcação e identifica características específicas de subpopulações por uma variedade de técnicas de marcação fluorescente.

Caracterização da Proteína Transmembrana da Vesícula Extracelular Única por Citometria de Nanofluxo

O presente protocolo fornece descrições detalhadas para o isolamento eficiente de vesículas extracelulares urinárias utilizando esferas magnéticas funcionalizadas. Além disso, engloba análises subsequentes, incluindo western blotting, proteômica e fosfoproteômica.

Isolamento Químico por Afinidade de Vesículas Extracelulares de Biofluidos para Análise Proteômica e Fosfoproteômica

Um protocolo detalhado é fornecido para limpeza e reutilização de tubos de ultracentrífuga de policarbonato para realizar o isolamento de vesículas extracelulares adequado para experimentos proteômicos.

Reutilização de Tubo de Ultracentrífuga de Policarbonato em Análises Proteômicas de Vesículas Extracelulares

Aqui, demonstramos o método de caracterização de vesículas extracelulares (EVs) coletadas de fluidos biológicos, como lágrimas e saliva, de seres humanos. O scanner usado neste método é capaz de detectar o fenótipo, tamanho e contagem total de partículas de EVs a partir de 1 μL da amostra.

Caracterização de Vesículas Extracelulares a partir de Fluidos Biológicos

Este vídeo mostra uma técnica eficaz para diferenciar e dissecando os vários semi-transparente das estruturas do corpo humano na pós vítreos olhos mortem.

Dissecção da Human Elements Corpo Vítreo para Análise Proteômica

Aprenda a isolar e caracterizar vesículas extracelulares derivadas de plasma humano para descoberta de biomarcadores cardiovasculares. Este protocolo utiliza análise proteômica por LC-MS/MS para identificar proteínas relevantes em doenças cardíacas.

Isolamento, caracterização e análise proteômica de vesículas extracelulares derivadas de plasma para descoberta de biomarcadores cardiovasculares (Vídeo) | JoVE Journal

Isolamento, caracterização e análise proteômica de vesículas extracelulares derivadas de plasma para descoberta de biomarcadores cardiovasculares

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