SW e TMV são suportados pelo NCRR NHLBI e no National Institutes of Health. BSK e HS são suportados pelo NINDS e NIGMS do National Institutes of Health.

Procedimentos experimentais O procedimento experimental (Fig. 2) consiste em quatro partes que são descritas em uma maneira passo a passo abaixo. Parte 1: subclonagem e expressão do C-terminal de P2X2 receptores. Nós expressamos o comprimento total C-terminal de receptores P2X2 em bactérias para identificar as proteínas do cérebro para que ele se liga. <ol><li> O C-terminal (resíduos 353-472) do receptor P2X2 (Fig. 1) foi amplificado por PCR, clonados em pGEX 4NT1 (GE Life Sciences) e verificadas com o seqüenciamento. </li><li> O plasmídeo recombinante foi transformado em E. Coli (BL21) para a expressão de proteínas recombinantes. </li><li> Stocks de glicerol de bactérias (E. coli BL21) contendo o P2X2 CT-GST plasmídeo foram raspadas com uma ponteira de pipeta estéril e inoculadas em 5 ml de Luria-Bertani (LB) de mídia com marcador seletivo adequado (50 g / ml ampicilina) e incubadas durante a noite em um agitador orbital em 37C. </li><li> As culturas cresceram durante a noite foram inoculadas em 250 ml de mídia LB com um antibiótico adequado (50 mg / ml ampicilina) e incubados em agitador orbital a 250 rpm por 2-3 horas a 37C, até a densidade óptica a 600 nm chegou ~ 0,6-0,7. </li><li> A cultura foi induzida com 1 mM de IPTG e ainda incubados a 37 ° C por 3 horas para a expressão da proteína. </li><li> A cultura foi então centrifugada a 5000 g por 15 min a 4C (1 ml da cultura foi salvo para analisar o nível de expressão e rotulados como &quot;induzida&quot;). Sobrenadante foi descartado eo pellet foi ressuspendido em 20 ml de solução salina Tris-EDTA (STE) tampão (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH 8,0). </li><li> A suspensão foi centrifugada a 5000 g por 15 min em um tubo falcon 50 ml. O sobrenadante foi descartado eo pellet semi-seco foi congelado a-70C durante a noite. </li><li> A-70C pellet foi ressuspendido em 40 ml de tampão de lise de gelo frio (2% (w / v) sarkosyl, 15 mg de lisozima, 150 mM NaCl, 50 mM Tris pH 7,5, 5 mM DTT e comprimido inibidor da protease completa) , ea suspensão foi incubada em gelo por 30 min. Durante esta incubação, 3 ml contas Glutationa-Sepharose 4B foram lavados com 20 ml de tampão fosfato salina (PBS) e 20 ml T-PBS (0,1% (v / v) tritonX-100 em PBS), seguida por PBS. As contas foram ressuspendidos em 1 ml de PBS. </li><li> A suspensão bacteriana foi freeze-descongeladas três vezes em nitrogênio líquido e sonicado (1s on / off, 30 microns por 3 min no gelo) e incubadas por mais 30 minutos com 4% (v / v) de triton X-100, 10 mM MgSO 4 e 2 mM ATP no gelo. </li><li> O lisado foi centrifugado a 20.000 g por 15 min a 4C e pellet foi descartado (a amostra de pellet e 1 ml do sobrenadante foi guardado para página SDS para testar as quantidades de proteínas no citoplasma e membranas). O sobrenadante foi incubado com as contas para 1 hr (ou noite) em 4C. </li><li> As contas eram girado em 500 g por 5 min eo sobrenadante foi descartado (amostra foi salvo para a fração não ligada). As pérolas foram lavadas cinco vezes com T-PBS (lavagens foram salvos para controles de lavagem). </li><li> As pérolas foram então ressuspendidas em PBS para dar 50% (w / v) O chorume e armazenados em geladeira a 4C até o uso. A concentração de proteína foi estimada pelo ensaio de Bradford (por instruções do fabricante). </li></ol> Parte 2: Preparação do lisados ​​cérebro inteiro P2X2 receptores são conhecidos por serem abundantemente expressa no cérebro 8. Na análise presente, buscou-se identificar as proteínas que interagem com receptores P2X2 de lisados ​​rato todo cérebro (Fig. 3). <ol><li> Cérebro de rato adulto foi colhida (animais foram sacrificados de acordo com um protocolo de UCLA IAACUC-aprovado e de acordo com o Guia do NIH para o Cuidado e Uso de Animais de Laboratório) e imediatamente lavados gelada PBS (3X). </li><li> Para lisar as células 10 ml (~ 5 vezes o peso úmido do cérebro colhida) de gelo lise frio contendo 150 mM NaCl, 50 mM Tris-HCl, pH 7,4, 10 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM Na 3 VO 4, 1 mM PMSF, 5 mg / ml de leupeptin, 1% (v / v) NP-40, e uma protease tablet cocktail inibidor foi adicionado ao cérebro colhida. </li><li> Tecido cerebral foi homogeneizado em gelo com um homogeneizador de Dounce vidro até uma mistura de consistência homogênea foi alcançado. </li><li> Lisado celular foi centrifugado a 2000 g por 5 minutos para remover as células ininterrupta. O sobrenadante foi coletado e girou novamente a 29.000 g por 60 minutos para remover organelas intactas e agregados membrana. </li><li> Imediatamente após a centrifugação o sobrenadante foi transferido para um tubo limpo. A concentração de proteína do cérebro inteiro lisado foi medida por ensaio de Bradford (normalmente ~ 15 mg / ml). </li><li> O lisado foi aliquotado e armazenado a-70C. </li></ol> Parte 3: Isolamento de P2X2 C-tail proteínas associadas Para identificar os parceiros de ligação de P2X2 CT-GST de lisados ​​cérebro inteiro, um pull down ensaio foi realizado usando CT-GST imobilizada em esferas de glutationa sepharose 4B como isca. Para controles, GST sozinho ligada a grânulos foi usado. <ol><li> Cérebro lisado foi contas devem ser pré-GST para 1 hora a 4C. O pellet foi descartado eo sobrenadante foi usado para análises posteriores. </li><li> Quantidades iguais de GST e P2X2 CT-GST (10 mg) obrigado a glutationa sepharose 4 contas B foram incubadas durante a noite com 100 g de proteínas solubilizadas precleared de rato lisado cérebro inteiro, com rotação de 4C no tampão de lise. </li><li> Contas foram centrifugada a 1000 g por 5 min eo sobrenadante foi descartado. </li><li> As pérolas foram lavadas uma vez com o tampão de lise e cozido em modificadas Laemli buffer [4% (w / v) SDS, 10% (v / v) 2-mercaptoetanol, 2% (v / v) de glicerol, 0,004% (w / v) de azul de bromofenol e 0,125 M pH 6,8 TrisCl] por 5 min. </li><li> As amostras foram girados em 5000 g por 5 min eo sobrenadante separado por 10% SDS-PAGE (Fig. 4). Géis foram corados com SYPRO gel de proteínas de Ruby mancha (de acordo com as instruções do fabricante) e visualizado sob luz ultravioleta. P2X2 CT-proteínas associadas foram comparados com aqueles recuperados usando GST nulo como isca. Quatro réplicas biológicas foram realizadas (ou seja, quatro cérebros e quatro downs puxar independentes). </li></ol> Parte 4: Identificação de proteínas. Proteínas separadas por eletroforese em gel foram excisados ​​do gel e identificados por espectrometria de massa 12. Nota: ausência de poeira ao longo do processo é fundamental para reduzir a contaminação de queratina. O experimentador deve usar uma máscara facial, rede de cabelo e luvas em todos os momentos e nunca toque na região gel de interesse sem o uso de luvas. <ol><li> Todas as bandas de proteínas visíveis recuperou a força para baixo P2X2 foram excisadas com uma lâmina de barbear limpo e picado (Fig. 4). Regiões correspondentes do gel na GST nulo pull down lane também foram excisadas da mesma forma sem levar em conta a coloração da banda (bandas específicas para a isca GST nulo também foram retirados e identificados) para garantir que a presença de proteínas abaixo do nível da sensibilidade a marcação não foi desperdiçada. </li><li> As peças gel foram incubadas com 200 mL de bicarbonato de amônio 50 mM em 50% (v / v) acetonitrila (ACN) e os tubos agitadas por 15 min em temperatura ambiente. Repita este passo de lavagem. </li><li> As peças foram desidratadas gel pela adição de 200 mL de acetonitrila (peças gel deve encolher e tornar-se opaco na cor). </li><li> Acetontrile foi removido e as peças em um gel seco speedvac para ~ 10 min. </li><li> As peças gel foram incubadas com 30 mL do recém-preparada 10 mM mM Tris DTT/10 (2-carboxi-etílico) fosfina cloridrato (TCEP) de volume suficiente para mergulhar as peças. Isso foi deixado por 30 min em banho-maria 56C. </li><li> Em seguida a solução de DTT foi substituído por 100 l de bicarbonato de amônio 500 mM em solução de acetonitrila 50% e as fatias de gel lavado com bicarbonato ammounium. </li><li> A solução de lavagem foi aspirado e 200 mL de acetonitrila adicionado ao desidratar o gel. </li><li> Acetonitrila foi então removido e 100 l preparada solução a 100 iodoacetamida mM foi adicionado em alquilação sulfhydryls livre. Esta etapa começou há 25 min à temperatura ambiente no escuro. </li><li> Solução iodoacetamida foi removido e as peças gel foram lavadas com 200 mL de bicarbonato de amônio 50 mM em acetontrile 50% (pH 8,0) por 2 min, enquanto vórtex. Esta etapa de lavagem foi repetido. </li><li> Após a remoção da solução de lavagem, as peças gel foram incubadas com 200 mL de acetonitrila que foi retirado por speedvac por ~ 10min. </li><li> Nesta etapa as peças gel está pronto para a digestão de tripsina. Pedaços de gel foram incubadas em 30 mL de solução de tripsina (20 concentração ng / mL de trabalho) e colocado em gelo por 10 min para o gel de ser bem inchado. Tripsina excesso foi removido e reidratados partículas de gel revestida com 30 mL com 50 mM de bicarbonato de amónio para mantê-los imersos durante a digestão que foi autorizado a prosseguir por 12 a 16 horas a 37C. </li><li> Cinco mL de 5% (v / v) ácido fórmico é adicionado para desativar a digestão com tripsina e prisão. </li><li> Tubos foram agitadas para ~ 15 min e centrifugado brevemente para levar o líquido até o fundo do tubo, que foi então transferido para um frasco limpo e rotulado ml 0,5. </li><li> ML 30 próxima de 0,1% (v / v) de ácido fórmico em acetonitrila 50% foi adicionado em pedaços gel que eles eram apenas cobertos seguido por vórtex duas vezes por 10-15 min, separado por centrifugação breve. Este passo foi repetido uma vez, substituindo o ácido fórmico e acetonitrilo entre as etapas. </li><li> O líquido final foi removido e toda a solução de extração combinados em um único frasco. Este volume foi reduzido para ~ 30 uL na speedvac. A amostra foi agora pronto para cromatografia de fase reversa nano-líquidos e identificação de proteínas por espectrometria de massa. Neste exemplo específico, esta etapa é realizada em um espectrômetro de massa Orbi-trap conjunto de ThermoFIsher (escolhido pela sua precisão massa elevada, ciclo de trabalho rápido para detecção da proteína e, geralmente, características operacionais robustos), embora outros modelos e instrumentos de fabricantes poderiam facilmente ser conjugada com a abordagem descrita para este ponto. </li><li> Os detalhes e critérios para análises de espectrometria de massa são brevemente apresentados. Todos os peptídeos foram separados por fase reversa nano-LC (Eksigent) e peptídeos foram carregados em uma coluna de fase reversa C18 a uma vazão de 3 mL / min. A fase móvel foi de 0,1% de ácido fórmico e ACN 2% da água; B da fase móvel foi de 0,1% de ácido fórmico e 20% de água em ACN. Peptídeos foram eluídos da coluna a uma vazão de 220 nl / min utilizando um gradiente linear de 5% para B B 50% mais de 90 min, e depois a B 95% mais de 5 min, e, finalmente, manter B constante de 95% por 5 min . Espectros foram adquiridos em data-dependente com o modo de Orbi armadilha utilizada para scans MS e LTQ para MS / MS scans. Peptídeos foram identificados através de pesquisa os espectros no banco de dados de rato IPI v.3.53 usando o algoritmo SEQUEST integrado no pacote de software Bioworks. Cada peptídeo preencheram os seguintes critérios: XCorr ≥ 2 (+1), ≥ 3 (2), ≥ 4 (+3), e DeltaCN&gt; 0,1. Todos os espectros utilizados para identificação de proteínas foram inspecionadas manualmente e sem proteínas aceitos quando menos de dois peptídeos foram identificados. Proteínas presentes apenas seguir cada um dos quatro downs P2X2 puxar e ausentes durante downs GST nulo pull foram considerados para análises posteriores. </li></ol><img src="/files/ftp_upload/1178/1178fig1.jpg" alt="figura 1" /> Figura 1. Representação esquemática de um receptor P2X2 subunidade. A. O desenho ilustra a topologia de uma subunidade do receptor P2X2. O domínio citosólico consiste em N e C termini. O C-terminal do receptor P2X2 (vermelho) foi usado como isca para puxar para baixo ensaio. B. seqüência de aminoácidos do receptor P2X2 C-terminal utilizado neste estudo. <img src="/files/ftp_upload/1178/1178fig2.jpg" alt="" /> Figura 2. Fluxograma e linha do tempo do protocolo utilizado para a expressão, purificação, puxar para baixo e identificação de proteínas. Mostramos o esboço do protocolo com a linha do tempo. Lisado de cérebro de rato adulto foi preparado fresco imediatamente antes do uso para a puxar para baixo os ensaios. <img src="/files/ftp_upload/1178/1178fig3.jpg" alt="" /> Figura 3. Representação esquemática da análise proteômica de binário P2X2 rede C-terminal no cérebro de ratos. O C-terminal de receptores P2X2 em fusão com GST proteína ligada a grânulos GST foi utilizada para puxar para baixo os ensaios. Lisado cérebro foi preparado e proteínas foram incubadas com as proteínas recombinantes imobilizado. Fracção livre foi lavado com o tampão de lise. Proteínas foram identificadas por espectrometria de massa. <img src="/files/ftp_upload/1178/Fig%204%20Singh%20et%20al.jpg" alt="" /> Figura 4. Identificação dos parceiros de ligação do C-terminal de P2X2 receptores. Sypro gel corado mostrando um espectro de proteínas putativas que interagem com o C-terminal dos receptores de fusão com GST (caixa azul). Controles pistas para GST sozinho (caixa amarela) e glutationa sepharose contas sozinhos também são mostrados. As setas indicam exemplos de bandas de originais que foram excisadas para posterior análise por espectrometria de massa.

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Canais iônicos são uma classe importante de proteínas da membrana integral. Eles contêm poros cheios de água que, seletivamente, permitir o movimento de íons para baixo suas gradientes eletroquímicos através da membrana plasmática. Ion portão canais entre os estados aberto e fechado. A etapa de propagação é desencadeada por transmissores (por exemplo, ATP) em caso de ligante P2X canais iônicos fechado, ou pode ser regulada por interações com outras proteínas. A última década testemunhou um aumento na ...

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		<th>Comment</th>
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<tr>
<td>Acetonitrile</td>
<td>Reagent</td>
<td>JT Baker</td>
<td>9829-02</td>
<td>&nbsp;</td>
<tr>
<td>Acrylamide</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0156</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1507-01</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
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<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
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<td>Dithiotritol (DTT)</td>
<td>Reagent</td>
<td>EMD</td>
<td>3860</td>
<td>&nbsp;</td>
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<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1474-01</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene Glycol tetraacetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
<td>&nbsp;</td>
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<td>Formic acid</td>
<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
<td>&nbsp;</td>
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<td>Glutathione Sepharose 4B beads</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-5132-01</td>
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<td>Hydrochloric acid (HCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1758</td>
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<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>15502</td>
<td>&nbsp;</td>
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<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I1149</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani (LB) Media</td>
<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
<td>&nbsp;</td>
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<td>Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L8511</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>Reagent</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
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<td>Magnesium Sulphate (MgSO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
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<td>Sodium Chloride (NaCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Flouride (NaF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Orthovanadate (Na3VO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P40</td>
<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
<td>&nbsp;</td>
<tr>
<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
<tr>
<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>170-3125</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N',N'-Tetramethylethylenediamine (TEMED)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9281</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

Canais de íon-ligante gated subjacentes comunicação sináptica no sistema nervoso 1. Nos mamíferos existem três famílias de ligante-gated canais: o laço cys, o glutamato-gated e os canais do receptor P2X 2. Em cada caso de ligação do transmissor leva à abertura de um poro através do qual os íons fluem seus gradientes eletroquímicos. Muitos ligand-gated canais também são permeáveis ​​aos íons de cálcio 3, 4, que têm a jusante de sinalização papéis 5 (por exemplo, regulação de genes) que podem exceder a duração da abertura do canal. Assim ligand-gated canais pode sinalizar em escalas de tempo amplo que vão desde alguns milissegundos até dias. Tendo em conta estes papéis importantes é necessário entender como ligante-gated canais iônicos em si são regulados por proteínas, e como essas proteínas podem sintonizar sinalização. Estudos recentes sugerem que muitos, senão todos, os canais podem ser parte de complexos de proteínas de sinalização 6. Neste artigo vamos explicar como identificar as proteínas que se ligam aos aspectos C-terminal do domínio receptor P2X2 citosólica. Receptores P2X são canais ATP-gated educação e composto por sete subunidades (P2X1-P2X7). Receptores P2X são amplamente expressa no cérebro, onde eles medeiam a transmissão sináptica excitatória e facilitação pré-sináptica da liberação de neurotransmissores 7. Receptores P2X são encontrados em células excitáveis ​​e não excitáveis ​​e mediar papéis-chave na sinalização neuronal, inflamação e função cardiovascular 8. P2X2 receptores são abundantes no sistema nervoso 9 e são o foco deste estudo. Cada subunidade P2X é pensado para dois segmentos possuem membrana estendida (TM1 e TM2) separados por uma região extracelular 7 e N intracelular e C termini (Fig 1a) 7. Subunidades P2X 10 (P2X1-P2X7) mostram homologia de seqüência de 30-50% no nível de aminoácidos 11. Receptores P2X conter somente três subunidades, que é o mais simples estequiometria entre os receptores ionotrópicos. O P2X2 C-terminal consiste de 120 aminoácidos (Fig 1b) e contém várias proteínas sites de consenso docking, apoiando a hipótese de que P2X2 receptor pode ser parte de complexos de sinalização. No entanto, embora várias funções foram atribuídas ao C-terminal de receptores P2X2 9 nenhum estudo descreveu os parceiros molecular que o casal para o lado intracelular desta proteína através do comprimento total C-terminal. Neste trabalho métodos que descrevem uma abordagem proteômica para identificar as proteínas que interagem com o comprimento total C-terminal de P2X2 receptores.

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Nós descrevemos um protocolo simples para identificar proteínas cerebrais que se ligam ao terminal de todo o comprimento C da ATP-gated P2X2 receptores. A extensão e aplicação sistemática desta abordagem para todos os receptores P2X deverá levar a uma melhor compreensão da sinalização do receptor P2X.

Proteômica para identificar proteínas Interagindo com P2X2 Canais Ligand-Gated Cation

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

proteomics-to-identify-proteins-interacting-with-p2x2-ligand-gated

Research

JoVE Journal

Biology

14.6K visualizações.  David Geffen School of Medicine, University of California, Los Angeles. Nós descrevemos um protocolo simples para identificar proteínas cerebrais que se ligam ao terminal de todo o comprimento C da ATP-gated P2X2 receptores. A extensão e aplicação sistemática desta abordagem para todos os receptores P2X deverá levar a uma melhor compreensão da sinalização do receptor P2X.

Vídeo: Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

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

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - um método para identificar em toda a Transcriptoma Sítios de Ligação de Proteínas RNA Binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Usando SCOPE para identificar potenciais Motivos regulamentação em Genes Coregulated

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantificação de Proteínas Usando Enriquecimento imunoafinidade Peptide Acoplado com Espectrometria de Massa

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

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 ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificado Sistema Yeast-Two-Hybrid para identificar proteínas Interagindo com o Fator de Crescimento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Reverter levedura Sistema de dois híbridos para identificar mamíferos Receptor Nuclear Resíduos que interagem com ligantes e / ou antagonistas

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Bottom-up e espingarda Proteômica para Identificar um Comprehensive Coclear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

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, ...

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.