Name: optimized protocol for the extraction of proteins from the human mitral valve
Uploaded: 2017-06-14T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve at JoVE.com

O Ministério da Saúde italiano apoiou este estudo (RC 2013-BIO 15). Agradecemos a Barbara Micheli por sua excelente assistência técnica.

Neste protocolo, os corações humanos são coletados durante a explicação multiorgânica (tempo de isquemia de 4-12 h, média 6 ± 2 h) de doadores de múltiplos órgãos excluídos do transplante de órgãos por razões técnicas ou funcionais, apesar dos parâmetros ecocardiográficos normais. Eles são enviados ao Banco de Tecidos Cardiovasculares de Milão, Monzino Cardiologic Center (Milão, Itália) para o banco das válvulas aórtica e pulmonar. Os folhetos mitrais posteriores não são utilizados para fins clínicos, portanto são coletados durante o isolamento da válvula aórtica e pulmonar após o consentimento informado dos parentes dos doadores. O tecido para transplante e pesquisa é coletado somente após o consentimento dos pais; Na folha de consentimento, eles autorizam (ou não) o uso do tecido cardíaco somente para pesquisa se não for adequado para uso clínico humano ( ou seja, problemas microbiológicos, funcionais e serológicos), seguindo as diretrizes do comitê de ética deMonzino Cardiologic Center. 1. Preparação da válvula mitral <ol><li> Colhe a válvula mitral humana o mais rápido possível após a explicação do órgão (tempo de isquemia de 4-12 h). </li><li> Em uma sala limpa, remova o coração do saco de transporte contendo uma solução fria (4 ° C) ( isto é, solução salina ou Eurocollins de média equilibrada ou Wisconsin). Coloque-o em um balde e coloque-o em um gabinete de biossegurança (fluxo de ar vertical de risco biológico, classe A, classificação boas práticas de fabricação (GMP)) para prosseguir com a preparação da válvula. </li><li> Coloque o coração sobre uma camada estéril descartável no armário. Usando um bisturi descartable estéril, corte o coração completamente, perpendicularmente ao seu eixo principal, no nível dos ventrículos esquerdo e direito, a cerca de 4 cm do ápice. </li><li> Mova a aorta ascendente e a artéria pulmonar para exibir o telhado do átrio esquerdo. </li><li> Com pinças esterilizadas autoclaváveis ​​e picaretas, corte ao redor da aurícula esquerda naTelhado do atrial esquerdo, visando a válvula mitral e permitindo a identificação do grande folheto mitral (anterior) e do pequeno folheto mitral (posterior). NOTA: As comissuras medulares antero-laterais e posteriores definem a borda do folheto anterior e a área posterior. </li><li> Usando tesoura esterilizada autoclavável e fórceps não traumáticos, dissecar o átrio esquerdo e a espessura da parede do ventrículo ao redor da circunferência de toda a valva mitral . </li><li> Identificar a continuidade da válvula mitro-aórtica. NOTA: O ventrículo esquerdo contém toda a válvula mitral e acordes. </li><li> Separe o folheto da válvula mitral anterior do folheto da válvula mitral posterior, cortando o folheto posterior ao longo da inserção com o ventrículo (comissura). </li><li> Lave o folheto posterior na solução salina. Corte o folheto em pequenos pedaços (&lt;1 cm 2 ) e envolva-os individualmente em papel alumínio. Feche-os com nitrogênio líquido. Cuidado: siga procedimentos de segurança organizacional ao usar nitrogênio líquido. <ol><li> Desinfecte a mesa do armário com uma solução de álcool isopropílico a 70% e uma solução de peróxido de hidrogênio a 6% no final do procedimento. </li></ol></li></ol> 2. Extração de proteínas <ol><li> Use fórceps para retirar a amostra armazenada em nitrogênio líquido e coloque-a imediatamente em gelo seco enquanto ainda está envolvido na folha de alumínio. Não deixe a amostra descongelar durante as transferências. </li><li> Antes da moagem, esfriar a argamassa de porcelana / zircónio e pilões de um sistema de moedor ( por exemplo, CryoGrinder) , juntamente com a amostra, colocando-os em um balão Dewar contendo nitrogênio líquido (~ 500 mL). Cuidado: siga procedimentos de segurança organizacional ao usar nitrogênio líquido. </li><li> Coloque a argamassa e pilões em uma caixa de poliestireno contendo gelo seco. Remova a amostra da folha de alumínio e coloque-a na argamassa. </li><li> Grind tEle provoca com o grande pilão contra a argamassa 15-20 vezes, usando a chave de fenda para girar o pilão. Misture a amostra com a ponta de uma espátula pré-congelada durante o processo de moagem. <ol><li> Repita com o pequeno pilão. </li></ol></li><li> Transfira a amostra de solo para um tubo previamente pesado ( por exemplo, tubo de centrífuga de 15 mL), invadindo o tubo, colocando-o sobre a argamassa e invadindo-os para mover a amostra para o tubo. Use uma espátula pré-refrigerada para recuperar todo o material da argamassa. <ol><li> Mantenha o tubo com a amostra em gelo seco para evitar a descongelação da amostra durante a transferência. </li></ol></li><li> Calcule o peso líquido da amostra. </li><li> Limpe a argamassa e os pilões após cada amostra e descontaminar-os por autoclave ou aquecê-los a 200 ° C por 2 h. </li><li> Transfira a amostra em pó do tubo de centrífuga para o tubo de vidro de um homogeneizador por inversão. </li><li> Adicione buffe de ureia filtradaR (8 M de ureia, 2 M de tioureia, 4% p / v de CHAPS, 20 mM de Tris e 55 mM de ditiotreitol) no tubo de vidro, 200 μL de tampão de ureia por cada 10 mg de tecido em pó. NOTA: A amostra de pó residual deixada no tubo da centrífuga pode ser recuperada usando parte do volume calculado de tampão de ureia. </li><li> Homogeneizar a amostra usando um agitador equipado com uma argamassa de vidro de borosilicato e um pilão de politetrafluoroetileno (PTFE). Pressione lentamente o pilão na amostra com um movimento de torção (1.500 rpm) 10 vezes. </li><li> Recupere o sobrenadante e transfira-o para um tubo de centrífuga limpo de 1,7 mL. Mais uma vez extraie a amostra restante com tampão de uréia fresco, adicionando metade do volume utilizado durante a primeira extração. </li><li> Repita o passo 2.10. </li><li> Recupere o sobrenadante e combine-o com o sobrenadante do passo 2.11. Coloque o sobrenadante combinado em um rotator de tubo durante 30 min. </li><li> Centrifugue o tubo por 30 min a 13.000 xg e 4 ° C. </li><li> Recupere o sobrenadante eD mede a concentração de proteína usando o teste de proteína de Bradford, de acordo com as instruções do fabricante. Armazene a amostra a -80 ° C até o uso. </li></ol>

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Um passo crítico deste protocolo é o uso de nitrogênio líquido para congelar a amostra e refrigerar o sistema de moagem. O uso de nitrogênio líquido evita a degradação biológica e permite o pulverização eficiente, mas requer treinamento específico para um manuseio seguro. Neste protocolo apresenta um sistema de moagem para moagem de amostras porque pequenas amostras são difíceis de recuperar de argamassa e pilão padrão. Neste caso, pequenas amostras se espalham como um pó fi...

Evidências recentes alteraram a compreensão dos papéis dos muitos mecanismos regulatórios que ocorrem após a síntese de mRNA. Na verdade, processos translacionais, pós-transcrição e proteolíticos podem regular a abundância e a função da proteína. O dogma - que diz que as concentrações de mRNA são proxies para as proteínas correspondentes, assumindo que os níveis de transcrição são o principal determinante da abundância de proteína - foi parcialmente revisado. De fato, os níveis de transcrição apenas prevêem parcialmente a abundância de proteína, sugerindo que os eventos pós-transcrição Ocorrem para regular as proteínas nas células 1 , 2 . Além disso, as proteínas determinam a função da célula e, portanto, ditar seu fenótipo, que pode sofrer mudanças dinâmicas em resposta a fatores autocrinos, paracrinos e endócrinos; Mediadores transmitidos pelo sangue; temperatura; Tratamento medicamentoso; E desenvolvimento da doençaMent. Assim, uma análise de expressão focada no nível de proteína é útil para caracterizar o proteoma e desvendar as mudanças críticas que ocorrem como parte da patogênese da doença 3 . Portanto, as oportunidades que a proteômica apresentar para esclarecer condições de saúde e doença são formidáveis, apesar dos desafios tecnológicos existentes. As áreas de pesquisa particularmente promissoras para as quais a proteômica pode contribuir: a identificação da expressão protéica alterada em qualquer nível ( ou seja, células inteiras ou tecido, compartimentos subcelulares e fluidos biológicos); Identificação, verificação e validação de novos biomarcadores úteis para o diagnóstico e prognóstico da doença; E, esperançosamente, a identificação de novos alvos de proteínas que podem ser utilizados para fins terapêuticos, bem como para a avaliação da eficácia e toxicidade da droga 4 . Capturar a complexidade deO proteoma representa um desafio tecnológico. As ferramentas proteômicas atuais oferecem a oportunidade de realizar análises em grande escala e de alto débito para identificação, quantificação e validação de níveis de proteína alterados. Além disso, a introdução de técnicas de fracionamento e enriquecimento, visando evitar a interferência causada pelas proteínas mais abundantes, também melhorou a identificação de proteínas, incluindo as proteínas menos abundantes. Finalmente, a proteômica foi complementada pela análise de modificações pós-tradução, que emergem progressivamente como importantes moduladores da função protéica. No entanto, a preparação da amostra e a recuperação de proteínas nos espécimes biológicos em análise continuam a ser as etapas limitantes no fluxo de trabalho proteômico e aumentam o potencial de possíveis armadilhas 5 . Na verdade, na maioria das técnicas de biologia molecular que devem ser otimizadas, os primeiros passos são homogeneização de tecidoLise de íons e células, especialmente durante a análise de proteínas de baixa abundância para as quais os métodos de amplificação não existem. Além disso, a natureza química das proteínas pode influenciar a sua própria recuperação. Por exemplo, a análise de proteínas altamente hidrofóbicas é muito desafiadora, porque elas se precipitam facilmente durante a focagem isoelétrica, enquanto as proteínas trans-membranas são quase insolúveis (revisada na Referência 5). Além disso, a variabilidade da composição do tecido cria uma barreira significativa ao desenvolvimento de um método de extração universal. Finalmente, porque quase todos os espécimes clínicos são de quantidade limitada, é essencial ativar a preparação de proteínas com recuperação máxima e reprodutibilidade a partir de quantidades mínimas de amostras 6 . Este trabalho descreve um protocolo otimizado para a extração de proteínas a partir da valva mitral cardíaca humana normal, o que representa uma amostra muito desafiadora para análise proteômica. A válvula mitral normal é um compEstrutura lex situada entre o átrio esquerdo e o ventrículo esquerdo do coração ( Figura 1 ). Ele desempenha um papel importante no controle do fluxo sanguíneo do átrio para o ventrículo, evitando o refluxo e garantindo o nível adequado de fornecimento de oxigênio para todo o corpo, mantendo um débito cardíaco adequado. No entanto, muitas vezes é considerado um tecido &quot;inativo&quot;, com baixa celularidade e poucos componentes, principalmente na matriz extracelular. Isso ocorre porque, em condições normais, as células intersticiais valvulares residentes (VICs) apresentam um fenótipo quiescente com baixa taxa de biossíntese de proteína 7 . No entanto, demonstrou-se que, em um estado patológico, o número de VICs no spongiosa aumenta e sua síntese protéica é ativada, juntamente com outras mudanças funcionais e fenotípicas 8 . Portanto, não é surpreendente que os dados mínimos disponíveis emA literatura se concentra no análise de válvulas mitrais patológicas 9 , 10 , nas quais o aumento do número de VICs ativados pode explicar o número relativamente alto de proteínas identificadas. Em conclusão, o presente protocolo pode servir para desenvolver a compreensão dos mecanismos patogênicos responsáveis ​​pelas valvopatias mitrais através do estudo dos componentes da proteína valvar mitral. De fato, uma maior compreensão dos processos patológicos subjacentes poderia ajudar a melhorar o manejo clínico das doenças valváticas, cujas indicações atuais de intervenção são em grande parte fundamentadas em considerações hemodinâmicas.

<table><tbody><tr><td>Saline solution</td><td></td><td></td><td>0.9 % NaCl</td></tr><tr><td>Eurocollins A</td><td>SALF</td><td>30874046</td><td>Balanced organ&#039;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td></tr><tr><td>Eurocollins B</td><td>SALF</td><td>30874022</td><td>Balanced organ&#039;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td></tr><tr><td>Wisconsin</td><td>Bridge life</td><td>RM/N 4081</td><td>Balanced organ&#039;s transport medium</td></tr><tr><td>Biohazard vertical flow air</td><td>Burdinola</td><td></td><td>Class A GMP classification</td></tr><tr><td>Dewar Flask</td><td>Thermo Scientific</td><td>Nalgene 4150-1000</td><td></td></tr><tr><td>Cryogrinder system</td><td>OPS diagnostics</td><td>CG 08-01</td><td>Grinder system containing mortars, pestles and screwdriver</td></tr><tr><td>Stainless steel forceps</td><td></td><td></td><td></td></tr><tr><td>Stainless steel spatula</td><td></td><td></td><td></td></tr><tr><td>Disposable sterile scalpel</td><td>Medisafe</td><td>MS-10</td><td></td></tr><tr><td>Stainless steel scissors</td><td></td><td></td><td>Autoclavable</td></tr><tr><td>Stainless steel picks</td><td></td><td></td><td>Autoclavable</td></tr><tr><td>Disposable sterile drap</td><td>Mon&amp;amp;Tex</td><td>3.307.08</td><td></td></tr><tr><td>Sterilizing solution with isopropyl alcohol</td><td></td><td></td><td>70% isopropyl alcohol</td></tr><tr><td>Sterilizing solution with hydrogen peroxide</td><td></td><td></td><td>6% hydrogen peroxide</td></tr><tr><td>Micropipette, 1 mL, with tips</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>VWR international</td><td>9278</td><td></td></tr><tr><td>1.7 mL centrifuge tubes</td><td>VWR international</td><td>PIER90410</td><td></td></tr><tr><td>Urea buffer</td><td></td><td></td><td>8 M urea, 2 M thiourea, 4 % w/v CHAPS, 20 mM Trizma, 55 mM Dithiotreitol</td></tr><tr><td>Urea</td><td>Sigma aldrich</td><td>U6504-1KG</td><td>To be used for Urea buffer</td></tr><tr><td>Thiourea</td><td>Sigma aldrich</td><td>T8656</td><td>To be used for Urea buffer</td></tr><tr><td>CHAPS</td><td>Sigma aldrich</td><td>C3023-5GR</td><td>To be used for Urea buffer</td></tr><tr><td>Dithiotreitol</td><td>Sigma aldrich</td><td>D0632-5G</td><td>To be used for Urea buffer</td></tr><tr><td>Syringe 50 mL</td><td>PIC</td><td></td><td>To be used to filter Urea buffer</td></tr><tr><td>0.22 &amp;micro;m filter</td><td>Millipore</td><td>SLGP033RB</td><td>To be used to filter Urea buffer</td></tr><tr><td>PFTE Pestle, 2 mL</td><td>Kartell</td><td>6302</td><td>Part of Potter-Elvehjem homogenizer</td></tr><tr><td>Borosilicate glass mortar</td><td>Kartell</td><td>6102</td><td>Part of Potter-Elvehjem homogenizer</td></tr><tr><td>Stirrer</td><td>VELP scientifica</td><td>Stirrer DLH</td><td>To be used for homogenization by Potter-Elvehjem</td></tr><tr><td>Bradford Protein assay</td><td>Bio-Rad laboratories</td><td>5000006</td><td></td></tr><tr><td>Tube rotator</td><td>Pbi International</td><td>F205</td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Polystyrene box</td><td></td><td></td><td></td></tr><tr><td>Dry ice</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td>For centrifugation of 1.7 mL centrifuge tubes at 13,000 x g</td></tr><tr><td>Freezer -80&amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>Precision balance</td><td></td><td></td><td></td></tr><tr><td>Autoclave</td><td></td><td></td><td>For sterilization</td></tr><tr><td>Cryogenic gloves for liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Professional forced ventilation and natural air convection oven</td><td></td><td></td><td>For sterilization</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma aldrich</td><td>P8340-5ML</td><td>100X solution</td></tr><tr><td>ProteoExtract Protein Precipitation Kit</td><td>Calbiochem</td><td>539180</td><td></td></tr><tr><td>RapiGest</td><td>Waters</td><td>186001861</td><td></td></tr><tr><td>Cytoscape</td><td>www.cytoscape.org</td><td>version 2.7</td><td>Software platform for Gene Ontology analysis</td></tr><tr><td>BiNGO</td><td>http://apps.cytoscape.org/apps/bingo</td><td>version 3.0.3</td><td>Plugin for Gene ontology analysis</td></tr><tr><td>AlphaB Crystallin/CRYAB Antibody</td><td>Novus Biologicals</td><td>NBP1-97494</td><td>Mouse monoclonal antibody against CryAB</td></tr><tr><td>Septin-11 Antibody</td><td>Novus Biologicals</td><td>NBP1-83824</td><td>Rabbit polyclonal antibody against septin-11</td></tr><tr><td>FHL1 Antibody</td><td>Novus Biologicals</td><td>NBP-188745</td><td>Rabbit polyclonal antibody against FHL-1</td></tr><tr><td>Dermatopontin Antibody</td><td>Novus Biologicals</td><td>NB110-68135</td><td>Rabbit polyclonal antibody against dermatopontin</td></tr><tr><td>Goat Anti mouse IgG HRP</td><td>Sigma aldrich</td><td>A4416-0.5ML</td><td>Secondary antibody for immunoblotting</td></tr><tr><td>Goat Anti rabbit IgG HRP</td><td>Bio-Rad laboratories</td><td>170-5046</td><td>Secondary antibody for immunoblotting</td></tr></tbody></table>

optimized protocol for the extraction of proteins from the human mitral valve

A análise do proteoma celular pode ajudar a elucidar os mecanismos moleculares subjacentes às doenças devido ao desenvolvimento de tecnologias que permitem a identificação e quantificação em larga escala das proteínas presentes em sistemas biológicos complexos. O conhecimento adquirido a partir de uma abordagem proteômica pode potencialmente levar a uma Melhor compreensão dos mecanismos patogênicos subjacentes às doenças, permitindo a identificação de novos marcadores diagnósticos e de doenças prognósticas e, espero, de alvos terapêuticos. No entanto, a válvula mitral cardíaca representa uma amostra muito desafiadora para análise proteômica devido à baixa celularidade em proteoglicano e matriz extracelular enriquecida com colágeno. Isso torna desafiador extrair proteínas para uma análise proteômica global. Este trabalho descreve um protocolo que é compatível com análises de proteínas subseqüentes, como proteômica quantitativa e imunotransferência. Isso pode permitir a correlação de dados relacionadosG expressão de proteína com dados sobre a expressão quantitativa de mRNA e análise imuno-histoquímica não-quantitativa. Na verdade, essas abordagens, quando realizadas em conjunto, levarão a uma compreensão mais abrangente dos mecanismos moleculares subjacentes às doenças, do mRNA à modificação da proteína pós-tradução. Assim, este método pode ser relevante para pesquisadores interessados ​​no estudo da fisiopatologia valvular cardíaca.

A extração e dissolução de proteínas no tampão de ureia é diretamente compatível com métodos proteômicos baseados em isolectrofocus (eletroforese bidimensional (2-DE) 11 e foco isoelétrico em fase líquida (IEF) 12 ) e com imunotransferência após diluição no tampão Laemmli 13 Contendo um coquetel inibidor de protease 14 . <p class="jove_content" fo:keep-together.within-page...

Watch this Scientific Journal Video about Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve at JoVE.com

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

A composição protéica da válvula mitral humana ainda é parcialmente desconhecida, pois sua análise é complicada pela baixa celularidade e, portanto, pela baixa biossíntese de proteínas. Este trabalho fornece um protocolo para extrair eficientemente proteínas para a análise do proteoma valvar mitral.

Protocolo otimizado para a extração de proteínas da válvula mitral humana

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

optimized-protocol-for-extraction-proteins-from-human-mitral

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

12.8K visualizações.  Centro Cardiologico Monzino IRCCS.  A composição protéica da válvula mitral humana ainda é parcialmente desconhecida, pois sua análise é complicada pela baixa celularidade e, portanto, pela baixa biossíntese de proteínas. Este trabalho fornece um protocolo para extrair eficientemente proteínas para a análise do proteoma valvar mitral. 

Vídeo: Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Proteína de captura por afinidade complexos a partir de células de mamíferos Cryomilled

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Bioquímica

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

Isolamento rápido do Mitoribosome de células HEK

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Dinâmica proteómica e miRNA análise de polissomos de coração de rato isolado após Langendorff perfusão

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

Eletroforese nativa clara/azul híbrida para a separação e análise de supercomplexos de cadeia respiratória mitocondrial

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Medição da Capacidade de Importação de Proteínas das Mitocôndrias Musculares Esqueléticas

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

<meta charset="utf8"></head><body>Isolamento mitocondrial de roedores para estudos bioenergéticos de alta qualidade

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>Autor em destaque: Descobrindo o papel do fosfato de cálcio mitocondrial na insuficiência cardíaca e bioenergética

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Isolamento mitocondrial robusto de tecido cardíaco de roedores 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Um protocolo para a identificação de interações proteína-proteína Baseado<sup&gt; 15</sup&gt; Etiquetagem N Metabólica, imunoprecipitação, Quantitativa Espectrometria de Massa e Affinity Modulação

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Biologia

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Isolamento de proteínas a partir da Embryonic Rato Coração Região Válvula Desenvolvimento

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Protocolo otimizado para a extração de proteínas da válvula mitral humana

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