Esta pesquisa foi financiada pelo fundo de arranque de Simon Fraser University.

1. Colheita Membranas <ol><li> Adicionar tampão hipotónico (10 mM Tris-HCl, 10 mM de KCl, 1,5 mM MgCl 2, pH 8,0) com o cocktail inibidor de protease para um agregado de células (~ 10 8 células) e incuba-se durante 15-30 minutos em gelo. Lyse as células por fazer passar a amostra através de agulhas de seringas (5-10 passagens) ou homogeneizar a amostra por um homogeneizador Dounce (15-30 AVC). Use um hemocitômetro e coloração azul de tripan para verificar a eficiência da lise. </li><li> Obter a fracção microssomal por uma centrifugação diferencial do ligado em 3000 gx 15 min e em seguida a 100.000 gx 2 hr. A primeira centrifugação obtém uma pelete que é a fracção nuclear, e a subsequente centrifugação do sobrenadante gera um segundo sedimento, que é a fracção microssomal que contém a membrana plasmática, bem como organelos ligados à membrana 8. O sobrenadante final é a fracção citosólica. Guarde as frações microssomais em freezer -80 ° C para mais analysé como se indica a seguir. </li></ol> 2. Dissolve, desnaturação e Digest Proteínas da Membrana <ol><li> Dissolve-se a fracção microssomal no tampão de desnaturação (Tris 40 mM, EDTA 10 mM, 10 mM de TCEP, 0,5% Rapigest, pH 8) e depois incubar a solução a 100 ° C durante 10 min. </li><li> Arrefece-se a solução aquecida até à temperatura ambiente, adicionar ultra-alta pureza ureia em pó a uma solução de 8 M de concentração final e incubar a amostra a 37 ° C durante 30 min. </li><li> Adicionar solução de estoque de iodoacetamida à amostra de 15 mM de concentração final, e incubar a solução no escuro durante 30 min à temperatura ambiente para alquilar os tióis livres na amostra. Adicionar solução de estoque de DTT para a amostra a uma concentração final de 10 mM e incuba-se a solução durante mais 10 min à temperatura ambiente para extinguir o excesso de iodoacetamida. </li><li> Dilui-se a solução obtida com 10x tampão de Tris 40 mM a pH 8, e, subsequentemente, adicionar tripsina à amostra a uma razão de 01:20 trypsin para a proteína total. Manter a reacção de digestão durante a noite num forno de 37 ° C para assegurar o fim da reacção. </li><li> Terminar a digestão por acidificação da solução de amostra para pH 1 com HCl, uma condição que também divide o detergente, ou seja Rapigest. Degradar o Rapigest a 37 ° C durante 1 hora, e remover a precipitação desenvolvido por centrifugação. </li><li> Limpar o sobrenadante que contém péptidos de amostra por um cartucho C-18 de extracção em fase sólida (SPE) e secar a amostra obtida por speedvac. </li><li> Realizar uma análise de SDS-PAGE de amostras antes e depois de digestão com tripsina para confirmar a eficiência da digestão. </li></ol> 3. Captura glicopéptido <ol><li> Dissolve-se os péptidos limpas no tampão (acetato de sódio 100 mM, pH 5,5) de acoplamento. Adicionar periodato de sódio na solução de péptido com uma concentração final de 10 mM durante 30 min no escuro, à temperatura ambiente. Isto irá oxidar os grupos cis-diol em glicanos de aldeídos, Que permitem que os glicanos a par com a resina através de química hidrazida. Extingue-se o excesso de periodato de sulfito de sódio a uma concentração final de 20 mM e pH 5 durante 10 minutos à temperatura ambiente. </li><li> Introduzir resina derivatizada com hidrazida para a solução de péptido com uma razão de 1:4 (resina de solução) para acoplar glicopéptidos à resina. Incubar a reacção a 37 ° C durante 1-2 dias com a rotação de ponta a ponta para o acoplamento completo. </li><li> Remover os péptidos não ligados por lavagem da resina duas vezes com 1 ml de cada um dos seguintes: água DI, 1,5 M de NaCl, 80% de metanol e acetonitrilo. Finalmente, lava-se a resina 3x com 1 ml de NH 4 HCO 3 a 100 mM, pH 8 para trocar o tampão do sistema de NH 4 HCO 3 a 100 mM. <ol><li> Recolhe-se o sobrenadante e as lavagens para a análise de péptidos não ligados (opcional). </li></ol></li><li> Soltar os N-glicopéptidos a partir da resina por PNGase F numa incubação durante a noite a 37 ° C com umn fim-de-final da rotação. Recolha os peptídeos liberados por centrifugação e lavagem acetonitrila 80%. Seca-se a solução obtida no Speedvac para análise por LC-MS. </li></ol> 4. Fraccionamento adicional (opcional) Para simplificar ainda mais a complexidade da amostra, fracionar as N-glicopeptídeos obtidos. Por exemplo, dissolver-se os péptidos secos em 10 mM de formato de amónia, pH = 3 com 20% de acetonitrilo e utilizar de permuta catiónica forte (SCX) de cromatografia para fraccionar os péptidos. Seca-se o eluente obtido, e, em seguida, analisar as fracções de péptido obtidas por inversão de fases LC-MS 8,17,18. 5. Limpeza de N-glicopeptídeos Lançado (Opcional) Se levantar preocupações para o potencial de contaminação dos péptidos, redissolver os péptidos secos em ácido fórmico a 0,1% e usar uma coluna SPE MCX para limpar ainda mais os péptidos antes de fase reversa A análise por LC-MS. <strong&gt; Nota: Os parâmetros de busca de banco de dados. Durante a clivagem selectiva de N-glicopéptidos fora da resina, PNGase F converte a asparagina ligada de N-glicanos de um ácido aspártico. Portanto, existe um deslocamento de massa de 0,9840 Da dos libertadas N-glicopéptidos. Para identificar com precisão estes péptidos, esta modificação pode ser adicionada para os parâmetros de pesquisa juntamente com modificações comuns, tais como o carbamidomethylation de cisteína e à oxidação do que a metionina.

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Os autores declaram que não têm interesses financeiros concorrentes.

Aqui apresentamos uma estratégia de captura de glycopeptide para perfilar as proteínas da superfície celular. O método pode ser aplicado para estudar as proteínas segregadas, tal como aqueles em sangue, assim como em outros fluidos corporais, ou em meios de cultura de células. O sucesso do método que se baseia na digestão completa de amostras; por conseguinte, uma caracterização de SDS-PAGE da eficiência da digestão é necessário, especialmente para a análise pela primeira vez ...

Proteínas da superfície da célula interagir com o ambiente extracelular e transmitir sinais a partir do exterior para o interior de uma célula. Assim, estas proteínas, incluindo proteínas de matriz extracelular, desempenham papéis críticos em todos os aspectos da biologia celular e fisiologia variando de proliferação, o crescimento, a migração, a diferenciação de envelhecimento e assim por diante. Proteínas de superfície de funcionar interagindo com outras células, proteínas e pequenas moléculas 1-3. Caracterização molecular de proteínas da superfície celular, é de grande interesse, não só para os biólogos, mas também para as empresas farmacêuticas, mais do que 60% ​​da droga são dirigidos a proteínas da superfície da célula 4. Espectrometria de massa em tandem (MS), com a sua sensibilidade superior, precisão e rendimento para a identificação de proteínas e peptídeos, tem sido uma ferramenta poderosa para estudos de proteômica globais 5,6. No entanto, as proteínas de superfície representam desafios significativos para proteômica baseada em MS, comoa maioria das proteínas de superfície existe em baixas quantidades, e com modificações pesados. As regiões que atravessam a membrana das proteínas de superfície de torná-los hidrófobos; este é especialmente o caso de proteínas transmembrana multipass. É, portanto, difícil de dissolver as proteínas de membrana em soluções aquosas, sem a ajuda de um detergente; no entanto, o uso de detergentes geralmente suprime o desempenho de HPLC e MS 1,7,8 na identificação de proteínas. Portanto, as proteínas da membrana foram mal caracterizado em proteômica baseada LC-MS diretos. A glicosilação é uma das modificações pós-tradução mais importantes e abundantes que ocorrem em proteínas da superfície das células 9. A enorme complexidade e heterogeneidade dos glicanos dificultar sinal de 10 MS &#39;peptídeos. No entanto, vários métodos proteomic usaram esta modificação única para enriquecer as proteínas de superfície e para remover as porções de açúcar a partir de proteínas antes da análise por LC-MS. Estes métodos incluem baseada em lectina de captura por afinidade e 11 à base de ácido de captura 12 ou químico bórico à base de hidrazida, bem como separações cromatográficas hidrofílicos 8,13. A remoção dos glicanos transforma as proteínas da membrana de proteínas e regulares drasticamente simplifica a caracterização MS. Porque glicosilação também ocorre em proteínas secretadas que têm alta solubilidade em contraste com a membrana proteínas, muitos métodos glycoproteomic são otimizados para proteínas solúveis, e tendem a ter menor seletividade glycopeptide e sensibilidade ao ser implantado para a membrana proteínas 8,14. Outros métodos também existe para o enriquecimento, em particular, as proteínas da superfície celular, tais como aqueles utilizando ultracentrifugação 15 e estratégias de rotulagem 16. Uma comparação detalhada entre o nosso método e outros métodos existentes para a caracterização de proteínas de membrana foi realizada recentemente 17, e os resultados indicaram que o método pode executar igualmente bem, se nãomelhor, do que todos os métodos de comparação proteómica membrana, mas com maior simplicidade. Para ajudar os pesquisadores a utilizar este método, detalhamos aqui um protocolo geral. Este método integra várias vantagens de estratégias glycoproteomics existentes e é concebido especificamente para glicoproteínas de membrana, no entanto, o método funciona igualmente bem para as proteínas secretadas. As características deste método incluem: 1) uma solubilização completa de proteínas de membrana, 2) um enriquecimento de glicopéptidos em vez de glicoproteínas para eliminar o potencial impedimento espacial quando se utiliza um substrato de captura de sólidos, 3) a utilização da química de hidrazida para formar ligações covalentes entre glicopéptidos e o substrato de captura, de modo a que os glicopéptidos ligados tolera lavagens rigorosas para alta glycoselectivity, e 4) a capacidade de realizar o processo de captura de todo em um tubo para a perda de amostra reduzida ea duração do procedimento encurtado. Depois de implementar este método para estudar uma variáveledade de amostras biológicas, incluindo células e tecidos, observou-se uma selectividade elevada (&gt; 90%) às glicoproteínas 8,17,18.

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646563</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodoacetamide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A3221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rapigest SF</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186001860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium periodate</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">311448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNGase F</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">P0704S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Affi-Gel Hz Hydrazide gel</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">153-6047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:199px;">Worthington Biomedical</td>
			<td style="width:119px;">LS02115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sep-Pak C-18 cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">WAT054955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor coctail</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:199px;">Amersco</td>
			<td style="width:119px;">568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium sulphite</td>
			<td style="width:199px;">Caledon</td>
			<td style="width:119px;">8360-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Invertase</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">I0408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alpha-1 trypsin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ribonulease B</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">R7884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Avidin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ovalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">C0755</td>
			<td></td>
		</tr>
	</tbody>
</table>

glycopeptide capture for cell surface proteomics

Proteínas de superfície celular, incluindo as proteínas da matriz extracelular, participam em todos os principais processos e funções celulares, tais como o crescimento, a diferenciação e proliferação. A caracterização detalhada destas proteínas fornece informações ricas para a descoberta de biomarcadores, a identificação do tipo de célula, e seleção-alvo da droga, bem como ajudando a avançar a nossa compreensão da biologia celular e fisiologia. Proteínas de superfície, no entanto, colocam desafios analíticos significativos, devido à sua inerente baixa abundância, alta hidrofobicidade, e pesadas modificações pós-traducionais. Aproveitando a glicosilação em proteínas de superfície predominante, apresentamos aqui uma abordagem de captura de glicopéptido de alto rendimento, que integra as vantagens de vários meios N-glycoproteomics existentes. Nosso método pode enriquecer os glicopeptídeos derivados de proteínas de superfície e remover seus glicanos para proteômica fáceis utilizando LC-MS. A N-glycoproteome resolvido compreende a informação de identidade e quantidade de proteína, bem como os seus locais de glicosilação. Este método foi aplicado a uma série de estudos em diversas áreas, incluindo o cancro, as células estaminais, e a toxicidade da droga. A limitação do método reside na fraca abundância de proteínas de membrana de superfície, de tal modo que uma quantidade relativamente grande de amostras é necessária para esta análise em comparação com estudos centrados em proteínas citosólicas.

Uma carta do procedimento experimental fluxo representativo é resumido na Figura 1. A rotulagem e as novas medidas de fracionamento são opcionais e os detalhes estão descritos em uma publicação recente 18. Uma outra opção é para analisar os péptidos não modificados, que não reagem com a resina. As vantagens da análise dos péptidos não modificados incluem a identificação potencial de peptídeos e proteínas não glicosiladas, tais como claudinas nas junções apertadas; uma van...

Watch this Scientific Journal Video about Glycopeptide Capture for Cell Surface Proteomics at JoVE.com

Glycopeptide Capture for Cell Surface Proteomics

As proteínas de superfície são biologicamente importantes e amplamente glicosilada. Apresentamos aqui uma abordagem de captura de glycopeptide para solubilizar, enriquecer e deglycosylate estas proteínas para análise proteômica baseada LC-MS fáceis.

Captura de glicopeptídeos de Superfície Celular Proteômica

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

glycopeptide-capture-for-cell-surface-proteomics

Research

JoVE Journal

Biology

11.2K visualizações.  Simon Fraser University.  As proteínas de superfície são biologicamente importantes e amplamente glicosilada. Apresentamos aqui uma abordagem de captura de glycopeptide para solubilizar, enriquecer e deglycosylate estas proteínas para análise proteômica baseada LC-MS fáceis. 

Vídeo: Glycopeptide Capture for Cell Surface Proteomics

Cell Capture Using a Microfluidic Device

Captura de célula usando um dispositivo micro

Biologia

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificação de Proteínas ortogonais Facilitado por um Tag Affinity Pequenas Bispecific

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimento cristalização de proteínas de membrana Usando o método Bicelle lipídico

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidez baseado Triagem Interação Extracelular (AVEXIS) para a detecção escalável de baixa afinidade Extracelular receptor-ligante Interações

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Captura de Espectrometria de Massa Compound - uma ferramenta poderosa para identificar novos c-di-GMP efetoras Proteínas

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Marcador de superfície celular Purificação Mediada de iPS Intermediários celulares a partir de um modelo Reprogramável mouse

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Não enzimática, Soro livre de Cultura de Tecidos de lesões mamárias pré-invasivas para a geração espontânea de Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Reação selecionada Monitoramento Espectrometria de Massa para Absolute Quantificação de Proteína

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Enriquecimento de Proteínas da Matriz Extracelular a partir de tecidos e digestão em péptidos para Análise de Espectrometria de Massa

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...