We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1. Preparação Lysate <ol><li> Crescer P. aeruginosa células em LB para a densidade óptica desejada. NOTA: Para obter orientação: usar ≈ 100 ml Cultura / amostra para culturas em fase estacionária e ≈ 500 ml cultur / sample para culturas em fase log (OD 600 nm = 0,5). </li><li> Pelete por centrifugação durante 20 min a 5.000 x g. </li><li> Ressuspender 0,5-1 g de sedimento em 1 ml de tampão de lise (6,7 mM de MES, HEPES 6,7 mM, NaCl 200 mM, Kac 6,7 mM, DDT 1 mM, pH 7,5) e adicionar inibidor de protease (mini completo, isento de EDTA), bem como DNaseI. </li><li> Lyse as células por três passagens através de uma célula de pressão francesa, a 20.000 psi (ver lista de materiais). </li><li> Ultra-centrifugação do ligado celular a 100.000 x g durante 1 h a 4 ° C. </li><li> Salve o sobrenadante (vá para o passo 2). </li><li> Lave o pellet com 1 ml 1x tampão de lise por pipetagem cima e para baixo. </li><li> Ultracentrífuga a 100.000 x g durante 1 h a 4 ° C. </li><li> Flash congelar o sedimento emazoto líquido e armazenar a -20 ° C até ser utilizado para a captura de proteínas membranares (ver etapa 3). </li></ol> 2. Remoção de nucleótidos livres c-di-GMP e Outros (Fração Solúvel Only) <ol><li> Lave uma coluna PD10 dessalinização (ver Lista de Materiais) com 10 ml de tampão de lise frio. </li><li> Verter o sobrenadante (≈ 1 ml) para a PD10, a fim de remover nucleótidos. </li><li> Elui-se com 4 ml de tampão de lise frio (500 ul passos). </li><li> Selecione as frações mais concentrados, como determinado pelo ensaio de Bradford, e reuni-los. </li></ol> (Apenas Fracção de Membrana) 3. pelota e ressuspensão Solubilização <ol><li> Ressuspender o sedimento em 500 a 1.000 ul de 1x tampão de captura (sem detergente) (ver Tabela 1). NOTA: O pellet é difícil para voltar a suspender. Aconselha-se a primeira pipeta cima e para baixo com uma pipeta para voltar a suspender aproximadamente o pellet, em seguida, usar uma seringa de 27 G para bem homogeneizar a solução. </li><li> Adicionar 1% (w / v) n-dodecil-β-D-maltopiranósido (DDM). </li><li> Incubar a 4 ° C durante pelo menos 2 horas (ou O / N) a uma roda rotativa. </li><li> Ultracentrífuga a 100.000 x g durante 1 h a 4 ° C. </li><li> Colha o sobrenadante. </li></ol> Medição da Concentração 4. Protein <ol><li> Medir a concentração de proteína pelo ensaio de Bradford (por ensaio BCA para a fracção de membrana). </li><li> Definir a concentração total de proteína de 10 mg / ml. </li></ol> 5. Captura <ol><li> Misturar 300 ug de proteína com 1 mM de PIB, GTP, ATP, CTP, e 20 ul de tampão de captura de 5x (100 mM de HEPES, 250 mM de KAc, MgAc 50 mM, 5% de glicerol, pH 7,5) (Tabela 1). Ajustar o volume da reacção para 100 ul com H 2 O. NOTA: o volume total inclui o volume do CDG-CC e c-di-GMP no caso do controlo de competição (ver passo 5.3 e Tabela 2). NOTA: Todos os experimentos foram realizados em 200 &amp;# 181; l de 12 tubos de tiras de PCR (Thermo Scientific). NOTA: para a fração de membrana, certifique-se de manter sempre a concentração DDM acima da concentração micelar crítica (0,01% w / v) até a etapa de solubilização de proteínas em 8 M ureia (Passo 7.1). </li><li> Incubar a 4 ° C durante 30 min numa roda rotativa. </li><li> Adicionar 10 uM CDG-CC (concentração final). NOTA: incluir um controle sem CDG-CC (referido como &quot;controle de talão&quot;), e um controle suplementado com 1 mM c-di-GMP (&quot;o controlo da concorrência&quot;) (ver Tabela 2). Nota: A concentração CDG-CC pode ser ajustado de 1 a 10? M. </li><li> Incubar a 4 ° C durante pelo menos 2 h (de N para a fracção S / membrana) sobre uma roda rotativa, no escuro. </li><li> Depois de uma breve centrifugação, de ligação cruzada pela activação do grupo reactivo do CDG-CC com luz UV durante 4 minutos, utilizando uma CaproBox (ver Lista de Materiais, λ = 310 nm, irradiância ≥10 mW / cm², a distância da fonte = 2 cm),. Nota: Retire a tampa das tiras cross-linking anterior. </li><li> Adicionar 25 ul de tampão de lavagem 5x (NaCl 5 M, Tris 250 mM, pH 7,5) e 50 uL de pérolas magnéticas de estreptavidina bem ressuspensos, homogeneizar suavemente. </li><li> Incubar a 4 ° C durante 1 hora numa roda rotativa. </li></ol> 6. Passos de lavagem NOTA: (Magnet: veja a lista de materiais). Comece com uma captura das esferas magnéticas na faixa tampa PCR, com o ímã. Em seguida, substituir a tira de PCR por um novo contendo a solução de lavagem seguinte. Remova o ímã e ressuspender as contas, e incubar 2 min. Spin down e substituir a tampa por uma nova tampa. <ol><li> As etapas de lavagem (fração solúvel apenas) <ol><li> Lavar 6 vezes em 200 ul de 1x tampão de lavagem. </li><li> Lavar uma vez em 200 ul de grau HPLC de H 2 O. </li><li> Lavar 6 vezes em 200 ul de 80% de acetonitrilo. </li><li> Lavar duas vezes em 200 ul de grau HPLC de H 2 O. </li></ol></li><li> As etapas de lavagem (membrafração ne apenas) <ol><li> Lavar cinco vezes em 200 ul de 1x tampão de lavagem + 0,1% de DDM. </li><li> Lavar duas vezes em 200 ul de 1x tampão de lavagem com 0,05% de DDM. </li><li> Lavar uma vez em 200 ul de tampão de lavagem 1x + 0,025% DDM. </li><li> Lavar uma vez em 200 ul de tampão de lavagem 1x + 0,0125% DDM. </li><li> Lavar 3 vezes, com 200 ul de 100 mM de ABC (bicarbonato de amónio, NH 4 CO 3) + 2 M de ureia. </li></ol></li></ol> 7. MS Preparação de amostras <ol><li> Ressuspender as contas (diretamente na tampa) em 20 ul ABC 100 mM (ABC mM 100 + 8 M uréia para a fração de membrana) e transferir para tubos de 1,5 ml. </li><li> Fracção de membrana só: incubar a 60 ° C durante 5 min, agitando a 500 rpm. </li><li> Adicionar 0,5 uL de 200 mM de TCEP (tris (2-carboxietil) fosfina) e incubar a 60 ° C durante 1 hora, agitando a 500 rpm. Arrefecer a 25 ° C. </li><li> Adicionar 0,5 mL de 400 mM frescamente preparado iodoacetamida e incubar a 25 ° C durante 30 min, agitando umat e 500 rpm no escuro. </li><li> Adicionar 0,5 uL de 0,5 M de N-acetil-cisteína e incubar a 25 ° C durante 10 min, agitando a 500 rpm. </li><li> Fracção de membrana só: adicionar 1 ul Lys-C e incubar a 37 ° C, O / N. </li><li> Adicionar 2 mg de tripsina e incubar O / N a 37 ° C, agitação a 500 rpm (envoltório em Parafilm para evitar a secagem). Observação: as amostras podem ser armazenadas a -20 ° C nesta fase. NOTA: fracção de membrana só: adicionar ABC 100 mM para ajustar a concentração de ureia para &lt;2 M antes da adição de tripsina. </li><li> Mexer um pouco abaixo dos tubos e recolher contas com o ímã. </li><li> Transferir o sobrenadante para um novo tubo de 1,5 ml (repetir este passo se grânulos são deixados). </li><li> Adicionam-se 5 ul de 5% de TFA (ácido trifluoroacético) + 1 ul de 2 M de HCl (15 mL de 5% de TFA + 5 ul de 2 M de HCl para a fracção de membrana). </li><li> Condição colunas C18 Microspin (The Nest Group, MA, EUA) com 150 ml de acetonitrila (spin 20 segundos a 2.400 rpm). </li><li> Equilibrate as colunas C18 duas vezes com 150 ul de 0,1% de TFA (20 segundos de centrifugação, 2.400 rpm). </li><li> Carregar a amostra e girar 2 min, 2000 rpm. </li><li> Recarregar o fluxo de passagem na coluna e repetir a etapa de fiação (2 min, 2000 rpm). </li><li> Lavar 3 vezes com 150 ul de 0,1% de TFA, 5% de acetonitrilo (20 seg, 2.400 rpm). </li><li> Tomar um novo tubo e eluir duas vezes com 150 ul de 0,1% de TFA, 50% de acetonitrilo (2 min, 2000 rpm). </li><li> Secam-se os péptidos em uma velocidade vac. </li><li> Ressuspender em 40 ul 98% de H 2 O, 2% de acetonitrilo, 0,15% de ácido fórmico. </li><li> Sonicate 20 sec (ciclo de pulso de 0,5, amplitude de 100%; veja a lista de materiais) e spin down 5 seg, 12.000 rpm (centrífuga de bancada). Vortex 10 seg, e spin down 5 seg, 12.000 rpm. Transferir num frasco de HPLC para análise por LC-MS / MS. </li><li> Congelar a -20 ° C. Observação: as amostras podem ser armazenadas a -20 ° C nesta fase. </li></ol> 8. LC-MS / MS Análise <ol><li> Executar um nano-LC (sistemas nano-LC) equed com uma coluna de RP-HPLC (75 mm x 37 cm) cheia com resina C18 (C18 mágica AQ 3 mm) usando um gradiente linear de 95% de solvente A (ácido fórmico a 0,15%, acetonitrilo a 2%) e 5% de solvente B ( 98% de acetonitrilo, ácido fórmico a 0,15%) para 35% de solvente B ao longo de 60 min a uma velocidade de fluxo de 0,2 mL / min. </li><li> Analisar os péptidos utilizando LC-MS / MS (dupla pressão LTQ-Orbitrap Velos espectrómetro de massa, ligada a uma fonte iónica de electropulverização). NOTA: O modo de aquisição de dados foi criado para obter uma alta resolução MS varredura na parte FT do espectrômetro de massa com uma resolução de 60.000 FWHM seguidos por varreduras MS / MS no ion trap linear dos 20 íons mais intensos. Para aumentar a eficiência de tentativas de MS / MS, o rastreio modus estado carregado foi activado para excluir não atribuído individualmente e carrega iões. Conluio dissociação induzida foi acionado quando o precursor ultrapassado contagem de íon 100. A duração da exclusão dinâmico foi ajustada para 30 seg. O tempo de acumulação de íons foi definido para 300 ms (MS) e 50ms (MS / MS). </li></ol> 9. Banco de Dados de Pesquisa <ol><li> Baixe o P. aeruginosa NCBI-banco de dados através da homepage do NCBI ( <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ). </li><li> Converta os espectros MS bruto em mascote arquivos genéricos (MGF), utilizando a ferramenta de conversão MassMatrix ( <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a> ). </li><li> Pesquisar neste arquivo mgf usando MASCOTE versão 2.3 contra o P. aeruginosa NCBI-banco de dados contendo a frente e entradas de proteína reversa de engodo. </li><li> Executar uma na digestão de tripsina silico após lisina e arginina (a não ser seguido por prolina) tolerando dois perderam clivagens na peptídeos trípticos totalmente. </li><li> Defina os parâmetros de pesquisa de banco de dados para permitir metioninas oxidada (15,99491 Da) como modificações variáveis ​​e carboxyamidomethylation (57,021464 Da) de resíduos de cisteína como a modificação fixa. Para MASCOTE nos perscrutaing scans de alta resolução, definir a tolerância massa precursor para 15 ppm e definir a tolerância massa fragmento de 0,6 Da. Finalmente, a proteína definida FDR para 1%. </li><li> Importe as pesquisas mascote do P. experimentos aeruginosa CCMS em Andaime (Proteomesoftware, versão 3), defina os parâmetros para obter uma proteína FDR perto de 1%, e extrair as contagens totais espectrais. </li><li> Para os resultados representativos apresentados neste trabalho, foi utilizado um teste t pareado para comparar a experiência com o controle de concorrência, e só consideradas hits com um valor p inferior a 0,1, e uma relação de contagem espectral acima de 2 (Experimento contagens espectrais / espectral competição controle contagens). </li><li> Os dados podem ser exportados em qualquer software de planilha para análise posterior. </li></ol> 10. livre-Label Quantificação <ol><li> Importe os arquivos brutos em software progênese LC-MS (Nonlinear Dynamics, versão 4.0). </li><li> Efetue o alinhamento LC-MS e detecção de recurso em Setti padrãoNGS. </li><li> Exportar os dados em formato de mgf progênese LC-MS. </li><li> Pesquisar os espectros de MS / MS usando o MASCOTE contra o NCBI P. banco de dados contendo aeruginosa frente e entradas de proteína reversa de engodo. </li><li> Importe os resultados da pesquisa de banco de dados em progênese LC-MS e mapear os peptídeos identificações para recursos MS1. </li><li> Para a avaliação de dados (cálculo de níveis de significância, dobram-change rácios) foi utilizado o script ProteinSQAnalysis de SafeQuant (limiar: valor q abaixo de 0,1, relação de contagem espectral acima de 2). </li></ol>

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The authors have nothing to disclose.

Um cuidado especial deve ser tomado em vários passos do protocolo. A concentração de proteína é um parâmetro crítico com uma concentração de 10 mg / ml de ser difícil de alcançar quando as células são cultivadas sob condições específicas de crescimento (por exemplo, biofilmes ou pequenas variantes de colónias). Assim, a re-suspensão da pelota deve ser realizada num pequeno volume de tampão de lise. As concentrações de proteína pode ser diminuída para 8 mg / ml. Comparado com o método pub...

c-di-GMP é um segundo mensageiro chave utilizados pela maioria das bactérias para controlar vários aspectos do seu crescimento e comportamento. Por exemplo, c-di-GMP regula a progressão do ciclo celular, motilidade e a expressão de exopolissacáridos e adesinas de superfície 2-4. Através da coordenação de tais processos c-di-GMP promove a formação de biofilme, um processo que está associado com infecções crónicas de uma variedade de bactérias patogénicas 5. c-di-GMP é sintetizado por enzimas chamadas ciclases diguanylate (PED) que abrigam um domínio GGDEF catalítico 4. Alguns PED possuem um site de inibidor que regula a atividade para baixo ciclase após ligação c-di-GMP. A degradação da proteína c-di-GMP é catalisada por duas classes distintas de fosfodiesterases (PDEs) ou albergando um EAL catalítica ou HD-GYP domínio 6,7. A maioria das proteínas efectoras conhecidos que se ligam directamente c-di-GMP pertencem a uma das três classes de proteínas: catalíticaGGDEF ou EAL domínios aliado inativos e domínios Pilz, pequenos interruptores moleculares que passam por mudanças de conformação após ligação c-di-GMP 8. PEDs, Pilz PDEs e proteínas estão bem caracterizados e os seus domínios pode ser prevista em silício de forma relativamente segura. Um interesse particular agora está focado na identificação de novas classes de efetores c-di-GMP. Alguns efetores c-di-GMP com diferentes motivos de ligação foram descritos recentemente, como o CRP / FNR Bcam1349 família de proteínas em Burkholderia cenocepacia ou o FleQ regulador de transcrição em P. aeruginosa 9,10. Além disso, riboswitches específico GMP-c-di foram recentemente identificados e mostrado para controlar a expressão de genes de uma forma c-di-GMP-dependente 11. Os motivos c-di-GMP ligação dos diferentes efetores são apenas mal conservada fazer previsões de bioinformática de tais proteínas difícil. Para resolver este problema, foi desenvolvido um método bioquímico, o qual se baseia na utilização de uma spe c-di-GMPCaptura espe- Composto combinada com a espectrometria de massa 1,12,13. Nós projetamos recentemente um romance trivalente c-di-GMP Captura Composto (CDG-CC, Figura 1) 1. Este andaime química é composta por: 1) um radical de c-di-GMP utilizado como isca para capturar c-di-GMP proteínas de ligação, 2) um grupo reactivo UV-fotoactivável usadas para reticular o CDG-CC para as proteínas ligadas e 3) uma biotina para isolar as proteínas capturadas usando esferas magnéticas revestidas com estreptavidina. O CDG-CC podem ser usadas para capturar directamente e especificamente efectores c-di-GMP partir de uma mistura complexa de macromoléculas como lisados ​​celulares. Composto de captura com base e abordagens químicas proteómica à base ter sido previamente relatado para ser aplicável a uma vasta gama de organismos, por exemplo, Caulobacter crescentus, Salmonella typhimurium enterica serovar e P. aeruginosa 1,14. Neste trabalho metodológico,nós fornecemos uma descrição em profundidade do procedimento CCMS utilizando extratos de P. aeruginosa como um exemplo. Este estudo estabelece CCMS como uma ferramenta poderosa e versátil para identificar bioquimicamente novos componentes envolvidos na sinalização molécula pequena.

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

Um progresso considerável foi feito durante a última década para a identificação e caracterização de enzimas envolvidas na síntese (ciclases diguanylate) e degradação (fosfodiesterase) do segundo mensageiro c-di-GMP. Em contraste, há pouca informação disponível sobre os mecanismos moleculares e componentes celulares através do qual essa molécula de sinalização regula uma gama diversificada de processos celulares. A maioria das proteínas efetoras conhecidos pertencem à família Pilz ou estão degenerados ciclases diguanylate ou phosphodiesterases que desistiram de catálise e adotaram a função efetora. Assim, para definir melhor a rede c-di-GMP celular numa vasta gama de bactérias, métodos experimentais são necessários para identificar e validar novos efectores para os quais fiável em predições silico falham. Recentemente, desenvolvemos um romance Captura Espectrometria de Massa Compound (CCMS) de base tecnológica como uma ferramenta poderosa paraidentificar e caracterizar bioquimicamente as proteínas de ligação c-di-GMP. Esta técnica tem sido relatada previamente para ser aplicável a uma vasta gama de organismos 1. Aqui nós damos uma descrição detalhada do protocolo que nós utilizamos para investigar tais componentes de sinalização. Como um exemplo, usamos a Pseudomonas aeruginosa, um agente patogénico oportunista, em que c-di-GMP desempenha um papel crítico no controle de biofilme e virulência. CCMS identificados 74% (38/51) dos componentes conhecidos ou previstos da rede c-di-GMP. Este estudo explica o procedimento CCMS em detalhe, e estabelece-lo como uma ferramenta poderosa e versátil para identificar novos componentes envolvidos na pequena molécula de sinalização.

Para identificar novos efectores c-di-GMP em P. aeruginosa foi utilizado sistematicamente CCMS para analisar as frações solúvel e de membrana de P. aeruginosa PAO1 estirpe a partir de uma cultura em fase logarítmica (OD600 = 0,5). Aqui vamos resumir e discutir os resultados representativos desta expedição de pesca. Foram utilizados quatro réplicas biológicas independentes. Para cada experimento, foram utilizadas duas concentrações CDG-CC diferentes (5 mm e 10 mm). Para investigar a...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

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

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 ...

capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Research

JoVE Journal

Biology

11.5K visualizações.  Biozentrum of the University of Basel. 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.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI preparação da amostra: o método de Camada Ultra Thin

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biologia

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

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

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 ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

A avaliação quantitativa do Lipidome fermento usando Electrospray Espectrometria de Massa de Ionização

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitina Yeast Membrana Baseado Two-Hybrid (MITO) do sistema: uma ferramenta poderosa para identificação de interações proteína-proteína

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Analisar grandes complexos de proteínas por espectrometria de massa estruturais

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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 ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Perfil de metiltransferases e Outros S-Adenosil- L Homocisteína-Proteínas de Ligação de captura por Espectrometria de Massa Compound (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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 ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amida de hidrogênio / deutério Exchange &amp; MALDI-TOF Análise Espectrometria de Massa de PAK2 Ativação

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Proteome Redox tiol Usando Isótopos Espectrometria de Massa Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...