Este trabajo fue financiado por la beca del Instituto de Biociencias de la Energía OO0G01.

El método se ejemplifica en OLIMP la hemicelulosa principales presentes en las paredes celulares de plantas dicotiledóneas, xiloglucano, utilizando un endoglucanasa como glycosylhydrolase. El método se demuestra con toda plántulas de Arabidopsis como una fuente de tejido de la planta. Material de la matriz extracelular y enzima se puede sustituir en función del análisis deseado utilizando el mismo procedimiento. 1. El aislamiento de la pared celular <ol><li> Cosecha de cinco 5 días de plántulas de Arabidopsis toda o la cantidad equivalente de su material deseado y transferirlos a un tubo de reacción 1,5 ml. Asegúrese de que el material se coloca en la parte inferior del tubo. </li><li> Agregue dos bolas de metal de 3 mm para el tubo de reacción en la parte superior de la muestra y la presión de congelación en nitrógeno líquido. </li><li> Triturar la muestra congelada con una molienda (2:30 min, 25 Hz). </li><li> Añadir 1 ml de etanol acuoso al 70% de la muestra. Eliminar las bolas de metal con un imán. Vortex a fondo. </li><li> Centrifugar la muestra a 14.000 rpm durante 10 minutos para que sedimenten los residuos insolubles en alcohol que contiene el material de la pared celular. </li><li> Retire con cuidado el sobrenadante por aspiración. Asegúrese de no perturbar el sedimento. </li><li> Añadir 1 ml de solución de cloroformo / metanol (1:1 v / v). Vortex a fondo para volver a suspender el pellet. </li><li> Centrifugar la muestra a 14.000 rpm durante 10 minutos y retirar con cuidado el sobrenadante por aspiración. Asegúrese de no perturbar el sedimento. </li><li> Secar la muestra utilizando un concentrador. </li></ol> 2. Solubilización de los oligosacáridos <ol><li> Para la solubilización del polisacárido particular en forma de sus oligosacáridos resuspender el pellet seco en 25μl de una solución amortiguadora. Para el formato de amonio endoglucanasa 50 mM, pH4.5, se utiliza. Añadir 0.2U de endoglucanasa. Vórtice de la suspensión y el líquido de girar. </li><li> Incubar la muestra durante 16 horas a 37 ° C en una incubadora, con agitación a 300rpm. </li><li> Eliminar el disolvente de la digestión (agua) utilizando un concentrador. </li></ol> 3. MALDI-TOF análisis de los oligosacáridos liberados <ol><li> BioRex MSZ 501 perlas de resina de intercambio catiónico se utilizan para eliminar las sales y la enzima de la muestra digerida. Estado de las cuentas mediante la colocación de una alícuota en una columna vacía y el lavado de los granos con grandes cantidades de agua. </li><li> Añadir 5-10 gotas BioRex de la muestra digerida y se secan. A continuación, añadir agua 10μl, por pequeñas cantidades de material de 5μl de agua puede ser utilizada. Sumerja las bolas en la solución de una breve gira el tubo. Incubar durante al menos 10 minutos a temperatura ambiente. </li><li> Lugar 2μl matriz (2,5-ácido hidroxibenzoico (DHB), 10mg/ml en agua) en una placa de MALDI objetivo. Se evapora el disolvente a vacío de la matriz que conduce a cristales homogéneos matriz química. </li><li> 2μl lugar de la solución de la muestra desalada en la parte superior de los cristales de la matriz en el plato blanco. Si desea detectar un gran número de muestras sólo seguir manchado por un máximo de 3 minutos. Este plazo se asegura de que la primera muestra manchas no está seco todavía. Espere a que un 2 minutos adicionales para asegurar una buena mezcla de re-disuelven la matriz y moléculas de la muestra de oligosacáridos en la última muestra de manchas. Luego, seque la placa de destino en el vacío. La mezcla de la muestra / matriz debe cristalizar en menos de 1 minuto para permitir la cristalización homogénea. </li><li> Lugar de destino en la placa de un espectrómetro de masas MALDI-TOF. La máquina debe estar en modo reflectron positiva con una tensión de aceleración de 20000V y un retraso de 350 nm, el rango de masas seleccionadas deben ser 500 a 3.000 Da (puede cambiar depende de los oligómeros de espera). Empiezan a disparar el láser y recoger 100-200 espectros por muestra, que se compilan para generar un espectro representativo medio (Figura 2A). Iones que representan oligosacáridos específicos se pueden identificar por su masa en carga (m / z) ratio. La intensidad de iones (altura máxima) de todos los iones de interés hay que añadir hasta 100%, resultando en la abundancia relativa de cada uno de oligosacáridos en la muestra (Figura 2B). </li></ol> 4. En el análisis in situ OLIMP OLIMP también se puede utilizar directamente sobre el tejido omitiendo los pasos de preparación de la pared. A modo de ejemplo etioladas hipocotilos de Arabidopsis como fuente tejido de la planta se utilizan. Una vez más, la endolucanasa se utiliza para determinar la estructura del xiloglucano hemicelulosas. Enzimas y material de tejido puede ser sustituido en función del análisis deseado utilizando el mismo procedimiento. <ol><li> Cosecha de una planta de semillero descoloridas y colocarlo en un plato blanco vacío MALDI y dejar que la semilla seca. </li><li> Añadir 0.5μl endoglucanasa (0.4U/μl, disuelto en formato de amonio 50 mM, pH4.5) en la plántula seca en la posición deseada. Asegúrese de que la caída de la enzima toca parte de la placa de destino. </li><li> Coloque la placa de MALDI objetivo en un recipiente cerrado con una humedad de saturación para evitar que la caída de la enzima se seca. Se incuba la placa en la cámara de un6 horas a 37 ° C. </li><li> Secar la placa MALDI objetivo con el tejido de la planta y la caída de la enzima al vacío. </li><li> Añadir 0.5μl matriz (DHB, 10 mg / l) en la parte superior de cada punto enzima seca. Deje reposar por 2 minutos. </li><li> Secar la placa MALDI objetivo al vacío. </li><li> Analizar las muestras con el MALDI-TOF. Lugar de destino placa con el tejido y el punto de enzima / matriz (s) en un espectrómetro de masas MALDI-TOF. La máquina debe estar en modo reflectron positiva con una tensión de aceleración de 20000V y un retraso de 350 nm, el rango de masas seleccionadas deben ser 500 a 3.000 Da (puede cambiar depende de los oligómeros de espera). </li><li> Empiezan a disparar el láser en los cristales / de la matriz siguiente muestra a los tejidos. Asegúrese de que no se alcanza el tejido en sí, como en este caso, el tiempo de vuelo de las moléculas se apagará. Reunir alrededor de 20 a 50 espectros por punto, que se compilan para generar un espectro representativo medio (Figura 3). Iones que representan oligosacáridos específicos se pueden identificar por su masa en carga (m / z) ratio. La intensidad de iones (altura de iones) pueden ser informados y todos los iones de interés hay que añadir hasta 100%, resultando en la abundancia relativa de cada uno de los oligosacáridos en la muestra. </li></ol> 5. Resultados representante Un ejemplo de un análisis de la OLIMP xiloglucano hemicelulosas presentes en plantas de Arabidopsis se muestra en la Figura 2. Debido a las diferencias de masa de los iones y la enzima conocida bien caracterizados (endoglucanasa) especificidad de los iones pueden ser asignados a determinadas estructuras de oligosacáridos (Figura 2A). Obviamente, isómeros estructurales no pueden ser distinguidos. El supuesto básico para la determinación de la abundancia relativa de los diversos oligosacáridos (Figura 2B) es que su espectrómetro de masas factor de respuesta es muy similar a los oligosacáridos. Como se muestra aquí, la cuantificación OLIMP es altamente reproducible. Sin embargo, tenga en cuenta que la solidez del método depende en gran medida la relación señal-ruido de los iones de oligosacáridos diferentes. Por ejemplo, la contaminación con sales o menores cantidades de oligosacáridos puede disminuir esa proporción. El análisis OLIMP en el propio tejido (análisis in situ) permite el estudio de áreas muy pequeñas y definidas y consiste en la preparación de muestras muy pequeñas. En el ejemplo que aquí se presenta (Figura 3) no cualitativo (iones mismo), pero las diferencias cuantitativas (variación en la intensidad de iones) se observaron entre los brotes y las raíces de tejidos de la plántula de Arabidopsis. Permutaciones de los OLIMP método de lo que podría dar lugar a tejidos &quot;imagen&quot;. <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="Figura 1" /> Figura 1. Diagrama de flujo del procedimiento OLIMP con plántulas de Arabidopsis como toda una planta. En primer lugar, el tejido se macera y material de la pared celular se prepara (foto modificada de Fujino et al. 10). A continuación, los oligosacáridos de un polisacárido de pared en particular son liberados con una hidrolasa específica. Por último, la abundancia relativa de los oligosacáridos solubles se determinan mediante MALDI-TOF espectrometría de masas. <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="Figura 2" /> Figura 2. Abundancia relativa de los oligosacáridos xiloglucano en plántulas de Arabidopsis etioladas según lo determinado por OLIMP. A) espectro Representante xiloglucano oligosacáridos de masa, cada ion representa una estructura de oligosacáridos específicos, isómeros estructurales no pueden ser distinguidos. B) diagrama de barras correspondiente para la determinación de la abundancia relativa de oligosacáridos. <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="Figura 3" /> Figura 3. En el análisis de OLIMP situ utilizando plantas de Arabidopsis etioladas como ejemplo. Cada gota de enzimas de digestión / (círculos de colores) pueden ser analizados de forma independiente y un espectro de masas correspondientes pueden ser obtenidos y analizados. <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="Figura 4" /> Figura 4. OLIMP espectro del xilano hemicelulosa obtenida por digestión de material de Miscanthus hoja con una xilanasa (Megazyme).

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El método que aquí se presenta OLIMP permite un análisis muy sensible y rápida de los polímeros presentes en las matrices extracelulares. OLIMP combina la liberación enzimática de oligómeros con posterior análisis MALDI-TOF. La generación de un espectro de MALDI-TOF toma menos de un minuto, por lo que OLIMP es adecuado para una amplia gama de aplicaciones, incluyendo de alto rendimiento estudios tales como pantallas de mutantes. OLIMP no se limita a los polisacáridos de plantas, pero potencialmente se pueden aplicar a una amplia gama de polímeros, sólo limitada por la disponibilidad de determinadas enzimas hidrolíticas. Sin embargo, una limitación de OLIMP es que la abundancia absoluta del polímero no se puede obtener. Como se mencionó antes OLIMP se puede utilizar para estudiar la estructura de una variedad de polisacáridos presentes en la matriz extracelular de una diversidad de especies. A modo de ejemplo, la Figura 4 representa un espectro OLIMP de la hemicelulosa en las principales especies de gramíneas, xilano. En este caso, el material de la pared celular derivado de la hierba templada Miscanthus fue digerido con una xilanasa.

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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>
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oligo mass profiling (olimp) of extracellular polysaccharides

El contacto directo de las células con el medio ambiente está mediada en muchos organismos por una matriz extracelular. Uno de los aspectos comunes de las matrices extracelulares es que contienen restos de azúcar en forma de complejo de glicoproteínas, proteoglicanos, y / o polisacáridos. Los ejemplos incluyen la matriz extracelular de las células animales y seres humanos que consiste principalmente de proteínas fibrilares y proteoglicanos o las paredes de polisacáridos basados ​​en células de plantas y hongos, y los proteoglicanos / glicolípidos paredes de las células base de bacterias. Todos estos glycostructures juegan un papel vital en la célula a la comunicación celular y celular con el medio ambiente y de señalización. Un ejemplo extraordinario complejo de una matriz extracelular está presente en las paredes de las células de plantas superiores. Su pared está hecha casi en su totalidad de los azúcares, hasta un 75% del peso seco, y se compone de los biopolímeros más abundantes presentes en este planeta. Por lo tanto, la investigación se lleva a cabo la forma de utilizar estos materiales de la mejor manera un recurso renovable de carbono-neutral para sustituir los productos petroquímicos derivados de combustibles fósiles. El principal desafío para la conversión de combustible sigue siendo la obstinación de las paredes a la degradación enzimática o química debido a la glycostructures únicos presentes en este biocompuesto único. A continuación, presentamos un método para el análisis rápido y sensible de glycostructures la pared celular vegetal. Este método de generación de perfiles Oligo Misa (OLIMP) se basa en la liberación enzimática de oligosacáridos a partir de materiales de la pared para facilitar glycosylhydrolases específicos y posterior análisis de las mezclas de oligosacáridos solubilizados con láser asistida por matriz de desorción / ionización tiempo de vuelo espectrometría de masas (MALDI-TOF/MS ) 1 (Figura 1). OLIMP requiere paredes de sólo 5000 células para un análisis completo, se puede realizar en el tejido mismo 2, y es susceptible de análisis de alto rendimiento 3. Mientras que la cantidad absoluta de los oligosacáridos solubilizado no se puede determinar por OLIMP la abundancia relativa de los iones de oligosacáridos diferentes pueden ser definidos a partir de los espectros de masas dando ideas acerca de la sustitución de patrón de los polisacáridos nativos presentes en la pared. OLIMP se puede utilizar para analizar una amplia variedad de polímeros de la pared, sólo limitada por la disponibilidad de enzimas específicas 4. Por ejemplo, para el análisis de polímeros presentes en las enzimas de la pared celular vegetal se encuentran disponibles para analizar el xiloglucano hemicelulosas con un xyloglucanase 5, 11, 12, 13, xilano con un 6,7 endo-β-(1-4)-xilanasa , o de polisacáridos pécticas utilizando una combinación de una poligalacturonasa y metilesterasa 8. Además, utilizando los mismos principios de OLIMP glycosylhydrolase e incluso actividades glicosiltransferasa se ​​puede controlar y decidir 9.

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

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

Una manera rápida se describe para obtener información sobre la estructura de los polisacáridos en una matriz extracelular. El método tiene la ventaja de la especificidad de glycosylhydrolases y la sensibilidad de la espectrometría de masas permite pequeñas cantidades de materiales para ser analizados. Esta técnica se puede adaptar para utilizarse directamente sobre el tejido en sí.

Oligo perfiles Misa (OLIMP) de polisacáridos 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 vistas.  University of California, Berkeley. Una manera rápida se describe para obtener información sobre la estructura de los polisacáridos en una matriz extracelular. El método tiene la ventaja de la especificidad de glycosylhydrolases y la sensibilidad de la espectrometría de masas permite pequeñas cantidades de materiales para ser analizados. Esta técnica se puede adaptar para utilizarse directamente sobre el tejido en sí.

Video: 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 preparación de la muestra: el método de ultra capa fina

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

Investigación

Biología

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.

Perfiles de anticuerpos por los sistemas de inmunoprecipitación luciferasa (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.

Perfiles de metiltransferasas y Otros S-Adenosil- L-Homocisteína proteínas que se unen mediante espectrometría de masas de captura compuesto (MCP)

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.

Perfiles de transcripción de los organismos unicelulares de los cardiomiocitos de ratón 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.

Creación de perfiles proteoma redox tiol Uso del etiquetado de espectrometría de masas

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.

Perfiles de Polímeros Glycan la pared celular vegetal utilizando 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 ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Análisis de la Traducción Iniciación Durante condiciones de estrés por polisoma Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

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.

Enriquecimiento de la Matriz Extracelular Las proteínas de los tejidos y la digestión en péptidos para el análisis de espectrometría de masas

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

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Un método fácil para la planta de perfiles de polisomas

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

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).

Perfilado Redox Celular Utilizando Microscopía de Alto Contenido

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