Este trabalho foi financiado pela concessão do Energy Biosciences Institute OO0G01.

O método é exemplificado OLIMP na hemicelulose principais presentes nas paredes celulares das plantas dicotiledôneas, xiloglucano, usando uma endoglucanase como o glycosylhydrolase. O método é demonstrado com toda mudas Arabidopsis como fonte de tecidos de plantas. Material de matriz extracelular de enzimas e pode ser substituído em função da análise desejada usando o mesmo procedimento. 1. Isolamento da parede celular <ol><li> Colheita cinco 5 dias de idade, todo mudas Arabidopsis ou a quantidade equivalente de seu material desejado e transferi-los em um tubo de reação 1,5 ml. Certifique-se que o material é colocado no fundo do tubo. </li><li> Adicione duas bolas de metal 3 milímetros ao tubo de reação em cima da amostra e encaixe-congelamento em nitrogênio líquido. </li><li> Moer a amostra congelada usando um moinho de bolas (2:30 min, 25Hz). </li><li> Adicione 1 ml de etanol 70% aquoso para a amostra. Retire as bolas de metal usando um ímã. Vortex completamente. </li><li> Centrífuga de amostra a 14.000 rpm por 10 min para sedimentar o resíduo de álcool insolúvel contendo o material da parede celular. </li><li> Remova cuidadosamente o sobrenadante por aspiração. Certifique-se de não perturbar o sedimento. </li><li> Adicione 1 ml de clorofórmio / metanol (1:1 v / v). Vortex cuidadosamente para ressuspender o sedimento. </li><li> Centrífuga de amostra a 14.000 rpm por 10 minutos e retire com cuidado o sobrenadante por aspiração. Certifique-se de não perturbar o sedimento. </li><li> Secar a amostra usando um concentrador. </li></ol> 2. Solubilização de oligossacarídeos <ol><li> Para a solubilização do polissacarídeo particular sob a forma de sua oligossacarídeos ressuspender o sedimento seco em 25μl de um tampão apropriado. Para o formiato de amônio 50 mM endoglucanase, pH4.5, é usado. Adicionar 0.2U da endoglucanase. Suspensão do Vortex e líquido de spin para baixo. </li><li> Incubar a amostra para 16h a 37 ° C em uma incubadora, agitando a 300rpm. </li><li> Remover o solvente da digestão (água), utilizando um concentrador. </li></ol> 3. MALDI-TOF análise dos oligossacarídeos liberados <ol><li> BioRex MSZ cação 501 contas resina de troca são usados ​​para remover sais e enzimas a partir da amostra digerida. Condição de as contas, colocando uma alíquota em uma coluna vazia e lavar as contas com grandes quantidades de água. </li><li> Adicionar contas de 10/05 BioRex para a amostra digerida e secas. Em seguida, adicione água 10μl, para pequenas quantidades de material de 5μl de água pode ser usada. Mergulhe as contas na solução por breves instantes, girando o tubo. Incubar por pelo menos 10 minutos em temperatura ambiente. </li><li> Local 2μl matriz (2,5-dihydroxybenzoic ácido (DHB), 10mg/ml em água) sobre uma placa de MALDI-alvo. Evaporar o solvente sob vácuo matriz levando a cristais químicos homogêneo matriz. </li><li> 2μl local da solução da amostra dessalinizada em cima dos cristais da matriz na placa-alvo. Se você quiser detectar um grande número de amostras apenas manter a mancha para um máximo de 3min. Este prazo garanta que a amostra spotted primeiro ainda não secou. Esperar por uma 2min adicionais para assegurar uma mistura completa de re-dissolvido matriz e moléculas da amostra de oligossacarídeos na amostra manchado passado. Em seguida, secar a placa-alvo sob um vácuo. A mistura amostra / matriz deve se cristalizar em menos de 1min para habilitar cristalização homogênea. </li><li> Coloque placa de alvo em um espectrômetro de massa MALDI-TOF. A máquina deve ser definida para o modo reflectron positiva com uma tensão de aceleração de 20000V e um atraso de 350 nm, a faixa de massa devem ser selecionados 500-3000 Da (pode mudar depende da oligômeros esperado). Começar a disparar o laser e recolher 100-200 espectros por exemplo, que são compilados para gerar um espectro médio representativo (Figura 2A). Íons representando oligossacarídeos específicos podem ser identificados pela sua massa mais razão de carga (m / z). As intensidades de íons (altura do pico) de todos os íons de interesse devem ser adicionados até 100%, resultando na abundância relativa de cada oligossacarídeo na amostra (Figura 2B). </li></ol> 4. Na análise OLIMP situ OLIMP também pode ser usado diretamente sobre o tecido omitindo qualquer etapas de preparação da parede. Como um exemplo de hipocótilos estiolados Arabidopsis como fonte de tecidos de plantas são usados. Mais uma vez, uma endoglucanase é usado para determinar a estrutura do xiloglucano hemiceluloses. Enzima e material de tecido pode ser substituído em função da análise desejada usando o mesmo procedimento. <ol><li> Colheita um mudas estioladas e coloque-a um prato vazio MALDI-alvo e deixe a seca das plântulas. </li><li> Adicionar 0.5μl endoglucanase (0.4U/μl, dissolvido em formiato de amônio 50 mM, pH4.5) na mudas secas na posição desejada. Certifique-se que a gota toca enzima parcialmente a placa de alvo. </li><li> Coloque a placa de MALDI-alvo em um recipiente fechado com umidade de saturação, para evitar que a queda da enzima seca. Incubar a placa na câmara para um6h a 37 ° C. </li><li> Secar a placa MALDI-alvo com o tecido vegetal ea queda das enzimas sob vácuo. </li><li> Adicionar 0.5μl matriz (DHB; 10mg / l) em cima de cada ponto da enzima secas. Deixe descansar por 2 minutos. </li><li> Secar a placa-alvo MALDI sob vácuo. </li><li> Analisar amostras com o MALDI-TOF. Coloque a placa com o tecido-alvo eo local da enzima / matriz (s) em um espectrômetro de massa MALDI-TOF. A máquina deve ser definida para o modo reflectron positiva com uma tensão de aceleração de 20000V e um atraso de 350 nm, a faixa de massa devem ser selecionados 500-3000 Da (pode mudar depende da oligômeros esperado). </li><li> Começar a disparar o laser para os cristais da matriz / amostra NEXT para o tecido. Certifique-se de não acertar o tecido em si, como no caso, o tempo de vôo das moléculas será desligado. Coletar cerca de 20-50 espectros por spot, que são compilados para gerar um espectro médio representativo (Figura 3). Íons representando oligossacarídeos específicos podem ser identificados pela sua massa mais razão de carga (m / z). As intensidades de íons (ion altura) pode ser relatado e todos os íons de interesse devem ser adicionados até 100%, resultando na abundância relativa de cada oligossacarídeos na amostra. </li></ol> 5. Resultados representante Um exemplo de uma análise OLIMP da xiloglucano hemiceluloses presentes em mudas de Arabidopsis é mostrado na Figura 2. Devido às diferenças de massa dos íons e da enzima conhecida bem caracterizado especificidade (endoglucanase) os íons podem ser atribuídos a estruturas específicas de oligossacarídeos (Figura 2A). Obviamente, isômeros estruturais não podem ser distinguidos. O pressuposto básico para a determinação da abundância relativa dos vários oligossacarídeos (Figura 2B) é que sua massa factor de resposta de especificações é muito semelhante para os oligossacarídeos. Como mostrado aqui, a quantificação OLIMP é altamente reprodutível. No entanto, observe que a robustez do método é altamente dependente do sinal para relações de ruído dos vários íons oligossacarídeo. Por exemplo a contaminação com sais ou menor quantidade de oligossacarídeos pode diminuir essa relação. A análise OLIMP sobre o tecido em si (na análise de situ) permite o estudo de áreas muito pequenas e definido e envolve a preparação da amostra muito pouco. No exemplo aqui apresentado (Figura 3) não qualitativa (íons mesma), mas diferenças quantitativas (variação nas intensidades ion) foram observadas entre parte aérea e raiz-tecido da plântula Arabidopsis. Permutações da OLIMP método, poderia, assim, levar a &quot;imagem&quot; do tecido. <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="Figura 1" /> Figura 1. Fluxograma do procedimento OLIMP utilizando mudas Arabidopsis inteiro como uma fonte de planta. Primeiro, o tecido é macerado e material da parede celular está preparado (foto modificada de Fujino et al. 10). Então oligossacarídeos de um polissacarídeo de parede especial são liberados através de um hidrolase específica. Finalmente, a abundância relativa dos oligossacarídeos solúveis são determinados utilizando MALDI-TOF espectrometria de massa. <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="Figura 2" /> Figura 2. Abundância relativa de oligossacarídeos xiloglucano em plântulas estioladas de Arabidopsis, conforme determinado pelo OLIMP. A) Representante xiloglucano espectro de massa oligossacarídeo; cada íon representa uma estrutura específica de oligossacarídeos, isômeros estruturais não podem ser distinguidos. B) diagrama de barras correspondentes para a determinação da abundância relativa de oligossacarídeos. <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="Figura 3" /> Figura 3. Na análise OLIMP situ utilizando mudas estioladas de Arabidopsis como exemplo. Cada gota de enzima / digestão (círculos coloridos) podem ser analisados ​​de forma independente e um espectro de massa correspondente podem ser obtidas e analisadas. <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="Figura 4" /> Figura 4. OLIMP espectro da xilana hemicelulose obtidos por digestão Miscanthus material da folha com uma xilanase (Megazyme).

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Não há conflitos de interesse declarados.

O método apresentado aqui OLIMP permite uma análise muito sensível e rápida de polímeros presentes nas matrizes extracelulares. OLIMP combina a liberação enzimática de oligômeros com a análise de MALDI-TOF subseqüentes. A geração de um espectro de MALDI-TOF leva menos de um minuto, daí OLIMP é adequado para uma ampla gama de aplicações, incluindo estudos de alto rendimento, como telas de mutantes. OLIMP não se restringe a polissacarídeos vegetais, mas pode potencialmente ser aplicada a uma ampla gama de polímeros, apenas limitada pela disponibilidade de enzimas hidrolíticas específicas. No entanto, uma limitação da OLIMP é que uma abundância absoluta de o polímero não pode ser obtida. Como mencionado anteriormente OLIMP pode ser usado para estudar a estrutura de uma variedade de polissacarídeos presentes na matriz extracelular de uma diversidade de espécies. Como exemplo, a Figura 4 representa um espectro OLIMP da hemicelulose importante em espécies de gramíneas, xilana. Aqui, material da parede celular derivada da temperadas grama Miscanthus foi digerido utilizando uma xilanase.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

oligo mass profiling (olimp) of extracellular polysaccharides

O contato direto das células para o meio ambiente é mediada, em muitos organismos por uma matriz extracelular. Um aspecto comum de matrizes extracelulares é que eles contêm metades açúcar complexo em forma de glicoproteínas, proteoglicanos e / ou polissacarídeos. Exemplos incluem a matriz extracelular dos seres humanos e células animais que consiste principalmente de proteínas fibrilares e proteoglicanos ou o polissacarídeo paredes à base de células de plantas e fungos, e os proteoglicanos / glicolipídeo paredes à base de células de bactérias. Todos estes glycostructures desempenham um papel vital na célula-célula de comunicação e de células-to-ambiente e de sinalização. Um exemplo extraordinário complexo de uma matriz extracelular está presente nas paredes das células de plantas superiores. Sua parede é feito quase inteiramente de açúcares, até 75% do peso seco, e consiste dos biopolímeros mais abundantes presentes no planeta. Portanto, a pesquisa é conduzida como utilizar esses materiais melhores como um recurso de carbono neutro renováveis ​​para substituir derivados petroquímicos de combustíveis fósseis. O principal desafio para a conversão de combustível permanece a recalcitrância de paredes à degradação enzimática ou química, devido à glycostructures única presente neste biocomposite único. Aqui, apresentamos um método para a análise rápida e sensível da planta glycostructures parede celular. Este método Profiling Massa Oligo (OLIMP) baseia a liberação enzimática de oligossacarídeos a partir de materiais de parede facilitar glycosylhydrolases específica e posterior análise das misturas de oligossacarídeos solubilizada usando matrix-assisted laser de dessorção / ionização de tempo de vôo espectrometria de massa (MALDI-TOF/MS ) 1 (Figura 1). OLIMP exige paredes de apenas 5.000 células para uma análise completa, pode ser realizada no próprio tecido 2, e é passível de high-throughput análises 3. Embora a quantidade absoluta do oligossacarídeos solubilizados não pode ser determinado por OLIMP a abundância relativa dos íons vários oligossacarídeos pode ser delineada a partir dos espectros de massa dando insights sobre a substituição de padrões do polissacarídeo nativo presente na parede. OLIMP pode ser usado para analisar uma grande variedade de polímeros de parede, limitada apenas pela disponibilidade de enzimas específicas 4. Por exemplo, para a análise de polímeros presentes na planta enzimas da parede celular estão disponíveis para analisar o xiloglucano hemiceluloses usando um xyloglucanase 5, 11, 12, 13, xilana usando uma endo-β 6,7 (1-4)-xilanase , ou para polissacarídeos pécticos utilizando uma combinação de uma poligalacturonase e um methylesterase 8. Além disso, usando os mesmos princípios de OLIMP glycosylhydrolase e até mesmo atividades glicosiltransferase podem ser monitorados e determinados 9.

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

Uma maneira rápida é descrito para obter insights sobre a estrutura de polissacarídeos em uma matriz extracelular. O método aproveita a especificidade do glycosylhydrolases ea sensibilidade da espectrometria de massa permitindo que pequenas quantidades de materiais a serem analisados. Esta técnica é adaptável para ser usada diretamente sobre o tecido em si.

Oligo Profiling Massa (OLIMP) de polissacarídeos extracelulares

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K visualizações.  University of California, Berkeley. Uma maneira rápida é descrito para obter insights sobre a estrutura de polissacarídeos em uma matriz extracelular. O método aproveita a especificidade do glycosylhydrolases ea sensibilidade da espectrometria de massa permitindo que pequenas quantidades de materiais a serem analisados. Esta técnica é adaptável para ser usada diretamente sobre o tecido em si.

Vídeo: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Profiling única célula transcricional dos cardiomiócitos de rato adulto

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profiling glicana de Polímeros da planta da parede celular usando Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Avaliação da Seletiva mRNA Tradução em células de mamíferos por polissoma Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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

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

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

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

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

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Redox Cellular Profiling Usando Microscopia de Alto Conteúdo

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Epigenoma célula tipo-específicas de criação de perfil

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...