<html> 1. Deglycosylation enzimática <ol><li> Use tubos de PCR para minimizar a perda de água devido à evaporação. Um conjunto de etiquetas de tubos de 1-7. </li><li> Descongele o buffer 10X G7, o buffer de glicoproteína 10X de desnaturação, e os 10% NP-40 e bata suavemente os tubos para misturar o conteúdo. Mantenha em temperatura ambiente. </li><li> Coloque os frascos contendo enzima no gelo. Tentar minimizar descongelar / congelar ciclos. </li><li> Dissolva o conteúdo do frasco hCGβ (150 mg) em 600 mL de dH 2 O e manter em gelo. </li><li> Prepare 1 ml de 1X tampão G7 diluindo o estoque de 10X em dH 2 O. </li><li> Diluir 0,5 mL de PNGase F em 25 mL tampão 1X G7 e manter em gelo. </li><li> Prepare a mistura exoglycosidase (mix EG), combinando cada 2 l de α-N-Acetylgalactosaminidase, α1-2 fucosidase, β1-3 Galactosidase e α1-3, 6 Galactosidase. </li><li> Configurar tubos PCR, conforme indicado: </li></ol>ak &quot;&gt; <table border="1"><tbody><tr><td> Tubo / amostra # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ (0.25mg/ml) </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> Glicoproteína 10X Tampão de desnaturação </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td></tr><tr><td> dH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li> Tapar os tubos, misture delicadamente e coloque notermociclador, feche a tampa e desnaturar as proteínas de incubação de 10 minutos a 94 ° C, seguido de um 4 segure C ° (usando tubos de PCR em um termociclador muito evita a evaporação em amostras de pequeno volume). </li><li> Remover os tubos de termociclador e centrífuga para remover qualquer condensação visível. </li><li> Adicionar os seguintes reagentes conforme indicado: </li></ol><table border="1"><tbody><tr><td> Amostra # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10% NP-40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> Tampão G7 10X </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5 &amp; mu; L </td><td> 2.5μl </td></tr><tr><td> PNGase F 01:50 dil. </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td></tr><tr><td> Mix Deglycosylation </td><td> - </td><td> - </td><td> 2μl </td><td> 2μl </td><td> - </td><td> 2μl </td><td> 2μl </td></tr><tr><td> Mix EG </td><td> - </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td></tr><tr><td> dH 2 O </td><td> 10μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> Vol reação total. </td><td> 25 l μ </td><td> 25 l μ </td><td> 25 l μ </td><td> 25 l μ &lt;/ Td&gt; <td> 25 l μ </td><td> 25 l μ </td><td> 25 l μ </td></tr></tbody></table><ol start="12"><li> Fechar os tubos PCR usando novas tampas (descartar os usados ​​desde que não se encaixam corretamente após um ciclo de incubação). </li><li> Misture os tubos batendo quatro vezes e depois girar o conteúdo para baixo. </li><li> Coloque os tubos no termociclador e incubar a 37 ° C por 4 horas depois esfriar as amostras a 4 ° C. </li></ol> 2. SDS-PAGE de amostras deglycosylated <ol><li> Prepare 130 mL de tampão de carregamento fresco 3X reduzindo SDS, adicionando 4 mL de DTT 1.25m. </li><li> Adicionar 12,5 l do tampão de carregamento preparado 3X reduzindo SDS para cada amostra. </li><li> Fechar os tubos com tampas novas e bata suavemente os tubos para misturar. </li><li> Incubar os tubos em um termociclador a 94 ° C por 5 minutos e depois arrefecer a 4 ° C. </li><li> Carga de 30 mL de cada amostra e 10 ml do marcador da proteína em um gel de 10-20% Tris-Glicina. Salvar o restante da amostra para a parte 3.1. 10μl de carga de proteína marcadora ColorPlus Prestained. </li><li> Electroforese do gel em 130 volts à temperatura ambiente até que a frente corante é perto da parte inferior do gel. </li><li> Quando o gel terminar a execução, remover o gel do elenco e colocá-lo em uma pequena caixa de plástico com bastante Coomasie mancha azul para cobrir o gel. </li><li> Manchar o gel por 1 hora com agitação suave. </li><li> Lave o gel três vezes durante 30 minutos em 50 ml de solução destain. </li><li> Gravar as imagens usando um transiluminador de luz branca ou scanner. Alternativamente, o gel pode ser secado entre folhas de papel celofane em um frame. </li></ol> 3. Pro-Q Emerald 300 para detecção de proteínas glicosiladas em géis SDS-PAGE <ol><li> Em paralelo com o gel em 2.1), coloque o restante das amostras em um gel de 10-20% Tris-Glicina. Carga 10 e mu; L da proteína marcadora ColorPlus Prestained. </li><li> Enquanto o gel está sendo executado dissolver o Pro-Q reagente Emerald com DMF e preparar o estoque Fix Wash, e oxidantes soluções para o Emerald Pro-Q 300 mancha seguindo o manual do produto fornecido com o kit. </li><li> Quando a eletroforese é concluída, remova o gel de elenco e colocá-lo em uma caixa de plástico. </li><li> Corrigir o gel pela adição de 100 ml da solução de Fix e deixá-lo durante a noite a temperatura ambiente com agitação suave. </li><li> Lavar o gel com 100 ml da solução de lavagem para 10 a 20 minutos em temperatura ambiente com agitação gentios. Repetir a lavagem com solução de lavagem fresca. </li><li> Oxidar os carboidratos, incubando o gel com agitação suave por 30 minutos em 25 ml de solução oxidante. </li><li> Lave o gel como descrito no passo 3.5. </li><li> Enquanto que o gel é lavar preparar fresco Pro-Q Emerald 300 mancha por adição de 500 mL do Pro-Q solução reagente Emerald 300 dissolvido na etapa 3,2-25 ml decoloração buffer fornecido no kit. </li><li> Mancha do gel pela adição de 25 ml da mancha preparada no passo 3.8 e incubar no escuro com agitação suave durante 90 a 120 minutos. </li><li> Repita as duas etapas de lavagem descrito no passo 3.5. </li><li> Gravar as imagens com um transiluminador UV, a 300nm. Use o marcador kDa 80, que é marcado com verde esmeralda Pro-Q, para sobrepor a imagem UV para uma imagem de luz branca do gel mostrando a escada prestained. </li><li> Compare as imagens do gel Coomasie manchada no passo 2,10 com o gel Emerald Pro-Q manchada a partir do passo 3.11. </li></ol> 4. Resultados representativos As mudanças na migração de proteína após deglycosylation enzimática são mostrados na Figura 2. Compare a amostra controle (painel A, faixa 1) com o tratamento F PNGase (remoção de N-glicanos, pista 2), e Mix Deglycosylation (PNGase F; além de endo e exoglycosidases para remover O-glicanas, pista 3). Sem reduçãoem tamanho é visto depois de digerir com glicosidases adicional (faixa 4). Além de uma mudança em massa, tornam-se mais nítidas bandas como glicanos são removidos. A banda rodando sob o marcador kDa 17 (asterisco) representa o (MW: 16 kDa) totalmente deglycosylated hCGβ polipeptídeo. Outras bandas poderiam derivar de deglycosylation incompleta ou das proteínas não-identificados vários presentes na amostra hCGβ (veja pista 1). Lanes 5-7 (controles) mostram as bandas correspondentes às glicosidases. A coloração da glicoproteína pelo verde esmeralda é mostrado no painel B. Este reagente oxida e manchas todos os glicanos presentes em uma molécula de proteína. Portanto, a intensidade do sinal diminui à medida que hCGβ é enzimaticamente deglycosylated (pista 1 a pista 4). O sinal residual em faixas 3 e 4 indicam a presença de motivos glicano, que são resistentes às enzimas utilizadas. O glicosidases adicional utilizado na pista quatro remover alguns resíduos de açúcar extra: a migração de proteínas é a mesma, masuma ligeira redução na intensidade de coloração pode ser visto. Metades açúcar resistentes não estavam presentes em todas as espécies de proteína: algumas bandas não foram detectados pelo verde esmeralda (ausente na imagem UV, entre parênteses), indicando que eles foram extensivamente deglycosylated. Dados adicionais de suporte à conclusão de que é heterogeneamente hCGβ glicosilada. A banda inferior na pista 2 (seta) é fraco na imagem verde esmeralda, enquanto a faixa superior na pista 2 é brilhante, indicando que muitos grupos glicanos ainda estão presentes. Esses dados dão suporte à conclusão de que hCGβ recombinante expresso em células de camundongo contém múltiplas glycoforms 9. Estes glycoforms diferentes são devido à heterogeneidade inerente de glicosilação, onde alguns polipeptídeos não receber um glicano em cada local consenso e / ou algum glicanos são estendidos, enquanto outros ainda sobre a mesma proteína não são. <img alt="Figura 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> Figura 1.Padrões típicos de glicosilação para secretada ou glicoproteínas da superfície celular. <img alt="Figura 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> Figura 2. Géis SDS-PAGE mostrando deglycosylation enzimática de hCGβ. Um painel mostra uma coloração azul, enquanto Coomasie Painel B mostra os resultados do Pro-Q Emerald 300 para a visualização de proteínas glicosiladas. Número da amostra: 1, controle hCGβ; 2, PNGase F digestão; 3, digestão Mix Deglycosylation; 4, Mix Deglycosylation mais exoglycosidases digestão, as amostras de 5-7 são os controles de reagentes (O-Glyc, ó glicosidase; GNA, β-N -Acetylglucosaminidase; fucosidase Gal / FUC, β1-3 Galactosidase, α1-3, 6 Galactosidase e α1-2; GalNA, α-N-Acetylglalactosaminidase; Neu, Neuraminidase; PNGase; F PNGase)

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Os autores são empregados por New England Biolabs, que produz muitos dos reagentes utilizados neste artigo.

O método descrito aqui, usando deglycosylation enzimática e SDS-PAGE pode fornecer informações valiosas sobre o estado de glicosilação de uma proteína de interesse, enquanto glicano-reagentes específicos facilitar a interpretação dos dados. Este protocolo destina-se para os estudos iniciais da glicosilação de proteínas e é particularmente adequada para secretoras e glicoproteínas da membrana de células de mamíferos: as enzimas escolhido, neste caso, especificamente remover todas as N-glicanos, e ou N-glicanos mais e os açúcares mais comuns ampliar e formar o núcleo de O-glicanas. Glicosidases têm a vantagem adicional de ser leve, em comparação com métodos químicos deglycosylation, preservando a integridade de ambos os açúcares e proteínas backbone. Para elucidar a taxa de ocupação (que aminoácidos são glicosilada), extensão de glicosilação, ou para determinar a estrutura fina de glicanos, técnicas mais sofisticadas, como mespectrometria de bunda, cromatografia líquida ou RMN são obrigatórios. Devido à sua simplicidade, vários passos neste protocolo pode ser ajustado, substituídos e / ou combinados para acomodar várias necessidades experimental. No entanto, a fim de obter resultados que podem ser claramente interpretado é importante para entender seus pontos fortes e limitações. Primeiro, a especificidade e pureza do glicosidases são cruciais: apenas enzimas bem caracterizados testado para ser livre de proteases e outras atividades de contaminação devem ser usados. Infelizmente, não há uma definição unidade padrão enzimático para glicosidases, o usuário deve determinar a substituição apropriada de acordo com as especificações do fabricante. Segundo, uma escolha cuidadosa de detecção é necessário: a) reagentes de coloração de proteínas são úteis somente se os resultados deglycosylation em uma mudança significativa na massa molecular. Um resultado tão claro como mostrado aqui nem sempre é obtida. Em outros casos, vimos mi anormalgração após deglycosylation (longe do peso previsto molecular, ou a migração ainda mais lento). Este fenômeno não é bem compreendida, mas pode-se dizer que qualquer alteração na migração evidências de que a proteína tem sido deglycosylated. B) detecção de anticorpos com Açúcar apresenta desafios únicos limitando a sua aplicabilidade. Tem sido muito difícil gerar geral anti-glicano anticorpos, que são geralmente levantadas contra um alvo glicano complexo, o que restringe seu uso. Além disso, vários monoclonal anti-glicano anticorpos exibir reatividade cruzada indesejada 10. C) As lectinas (proteínas com afinidade intrínseca de açúcar) são adequados para a detecção de açúcar, mais eles dão uma visão sobre a estrutura de glicano. No entanto, nem todos eles têm uma especificidade estreita, e muitos são apenas parcialmente caracterizada (o que significa que eles poderiam ter afinidades desconhecido). Como conseqüência coloração lectina positivo fornece uma indicação, não uma prova da presença de um soldadoven açúcar. d) kits rotulagem química (com base na oxidação periodato de açúcares) são o método de escolha para manchar todas as glicoproteínas, e, portanto, bem adequado a seguir deglycosylation. Ao processar uma amostra desconhecida, é uma boa prática para incluir controles glicoproteína. Fetuína é um N prontamente disponível - e O-glicoproteína, gonadotrofina coriônica (ambas subunidades) também é uma boa escolha. Albumina de soro bovino (BSA) pode ser usado como controle negativo. No entanto, deve notar-se que algumas proteínas não-glicosiladas reagir um pouco com o Emerald Pro-Q 300, especialmente quando usado em altas concentrações. Não-glicosilada padrões de peso molecular, tais como o marcador de proteína prestained utilizado neste protocolo, têm a vantagem de exibir faixas afiadas. Só por acaso é uma dessas proteínas (80 kDa) reativa para pró-Q Emerald 300. Portanto, o usuário pode querer executar uma escada padrão glicoproteína vez, como Candy Cane-de Invitrogen. Finalmente, detecção em gel simples de glicoproteínas nucleocytosolic (que são modificados com um único O-GlcNAc) é possível com o uso de um anticorpo monoclonal, junto β-N-Acetylglucosaminidase para o controle de especificidade 11. A descrição desta técnica está além do escopo deste artigo, mas deve ser mencionado como glicosidases são ferramentas úteis para o estudo de muitas outras glicoproteínas e glicoconjugados presentes nas células. Um tratamento abrangente de todas as formas conhecidas de glicosilação pode ser encontrado na segunda edição do &quot;Essentials of Glycobiology&quot;; on-line gratuito, disponível no NCBI Bookshelf (ID Bookshelf: NBK1908; PMID: 20301239) 12.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Β gonadotrofina coriônica humana </td><td> Sigma Aldrich </td><td> C6572 </td><td></td></tr><tr><td> PCR Tubes </td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR Termociclador </td><td> Applied Biosystems </td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> P0704 </td><td> Fornecido com buffers </td></tr><tr><td> Mix Deglycosylation proteína </td><td> New England Biolabs </td><td> P6039 </td><td> Fornecido com buffers </td></tr><tr><td> α-N-Acetylglalactosaminidase </td><td> New England Biolabs </td><td> P0734 </td><td> Fornecido com tampão </td></tr><tr><td> α1-2 fucosidase </td><td> New England Biolabs </td><td> P0724 </td><td> Fornecido com tampão </td></tr><tr><td> α1-3, 6 Galactosidase </td><td> New England Biolabs </td><td> P0731 </td><td> Fornecido com tampão </td></tr><tr><td> β1-3 Galactosidase </td><td> New England Biolabs </td><td> P0726 </td><td> Fornecido com tampão </td></tr><tr><td> Tampão 10X G7 (fosfato de sódio 500 mM) </td><td> New England Biolabs </td><td> --- </td><td> Fornecido com PNGaseF ou Mix Degl </td></tr><tr><td> Tampão de desnaturação Glicoproteína 10X (5% SDS, 400 mM DTT) </td><td> New England Biolabs </td><td> --- </td><td> Fornecido com PNGaseF ou Mix Degl </td></tr><tr><td> 10% NP-40 </td><td> New England Biolabs </td><td> --- </td><td> Fornecido com PNGaseF ou Mix Degl </td></tr><tr><td> 3X SDS loading buffer (187,5 mM Tris-HCl pH 6,8, SDS a 6%, glicerol 30%, brom 0,03%azul ophenol) </td><td> New England Biolabs </td><td> B7703 </td><td> Fornecido com 1.25m TDT </td></tr><tr><td> ColorPlus Prestained marcador Protein </td><td> New England Biolabs </td><td> P7709 </td><td></td></tr><tr><td> 10-20% Tris-Glicina Multigel </td><td> Cosmo Bio Co. </td><td> DCB-414893 </td><td></td></tr><tr><td> Unidade de Eletroforese cassete </td><td> Cosmo Bio Co. </td><td> DCB-303111 </td><td></td></tr><tr><td> Eletroforese EPS Fonte de alimentação 301 </td><td> GE Healthcare </td><td> 18-1130-01 </td><td></td></tr><tr><td> Pro-Q Emerald Kit Glicoproteína 300 Stain </td><td> Invitrogen </td><td> P-21857 </td><td></td></tr><tr><td> Brilliant Blue R </td><td> Sigma Aldrich </td><td> B0149 </td><td></td></tr><tr><td> AlphaImager HP Sistema </td><td> Célula de Biociências </td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glicosilação, adição de açúcares ligados covalentemente, é uma modificação pós-translacional de proteínas importantes que podem afetar significativamente processos como adesão celular, o tráfico molecular, depuração e transdução de sinal 1-4. Em eucariotos, as modificações mais comuns de glicosilação na via de excreção são adições em consenso resíduos asparagina (N-linked), ou em serina ou resíduos de treonina (O-linked) (Figura 1). Início do N-glicano síntese é altamente conservada em eucariotos, enquanto o produto final pode variar muito entre diferentes espécies, tecidos ou proteínas. Alguns glicanos não haverá alterações (&quot;alta manose N-glicanos&quot;) ou são tratados posteriormente no Golgi (&quot;complexo N-glicanos&quot;). Uma maior diversidade é encontrada para O-glicanas, que começam com um comum N-acetilgalactosamina resíduos (GalNAc) em células animais, mas diferem em organismos inferiores 1. ent &quot;&gt; A análise detalhada da glicosilação de proteínas é um campo em si mesmo e exige muitos recursos e expertise para executar corretamente. No entanto uma variedade de enzimas que removem disponíveis açúcares (glicosidases) torna possível ter uma idéia geral do estado de glicosilação . uma proteína em uma configuração padrão de laboratório Aqui ilustramos o uso de glicosidases para a análise de uma glicoproteína modelo. recombinante gonadotrofina coriônica humana beta (hCGβ), que carrega dois N-glicanos e quatro O-glicanas 5 A técnica requer apenas simples instrumentação e consumíveis típica, e pode ser facilmente adaptado para a análise de amostras glicoproteína múltiplas. Várias enzimas pode ser usado em paralelo para estudar uma glicoproteína. PNGase F é capaz de remover quase todos os tipos de N-glicanos ligados 6,7. Para O-glicanas, não há enzima disponíveis que podem decompor um oligossacarídeo intacto de thbackbone e proteína. Em vez disso, O-glicanas são aparados por exoglycosidases a um núcleo pequeno, que depois é facilmente removido por O glicosidase. O Mix Deglycosylation Protein contém PNGase F, O glicosidase, Neuraminidase (sialidase), β1-4 Galactosidase e β-N-Acetylglucosaminidase. Ele é usado para remover simultaneamente N-glicanos e alguns glicanos O-8. Finalmente, o Mix Deglycosylation foi suplementada com uma mistura de exoglycosidases outros (α-N-Acetylgalactosaminidase, α1-2 fucosidase, α1-3, 6 Galactosidase e β1-3 Galactosidase), que ajudam a remover monossacarídeos de outra forma resistente, que podem estar presentes em alguns O-glicanas. SDS-PAGE/Coomasie azul é usado para visualizar diferenças na migração de proteína antes e após o tratamento glicosidase. Além disso, um açúcar específico método de coloração, ProQ Emerald-300, mostra o sinal diminuído como glicanos são successively removido. Este protocolo é projetado para a análise de pequenas quantidades de glicoproteína (0,5 a 2 mg), embora deglycosylation enzimática podem ser ampliados para acomodar grandes quantidades de proteína, conforme necessário.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Usando glicosidases específicas para remover a partir de açúcares glicoproteínas seguido por SDS-PAGE é um método valioso para detectar modificações glicano em amostras de proteína e é uma boa escolha para estudos glycobiology inicial. Seguintes alterações deglycosylation podem ser detectados como mudanças na mobilidade gel ou através da coloração com glicano reagentes sensíveis.

Identificação e caracterização de glicosilação de proteínas específicas usando endo-e exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

36.4K visualizações.  New England Biolabs. Usando glicosidases específicas para remover a partir de açúcares glicoproteínas seguido por SDS-PAGE é um método valioso para detectar modificações glicano em amostras de proteína e é uma boa escolha para estudos glycobiology inicial. Seguintes alterações deglycosylation podem ser detectados como mudanças na mobilidade gel ou através da coloração com glicano reagentes sensíveis.

Vídeo: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificação de complexos de proteínas com proteômica quantitativa em S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biologia

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery Fase Líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificação de parceiros de proteína Interagir utilizando Tandem purificação por afinidade

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Microarray de anticorpos quimicamente bloqueada para Multiplexed perfil de alta capacidade de glicosilação proteína específica em amostras complexas

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Método para o isolamento e identificação de mRNAs, microRNAs e componentes proteicos de complexos de ribonucleoproteínas de extratos celulares utilizando RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Geração, purificação, caracterização e de Cell-invasivos espécies de proteína Disc1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pirosequenciamento de Identificação Microbiana e Caracterização

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.