Damos las gracias al Fondo para UAMS Proteómica para el apoyo de espectrometría de masas. Los fondos para este proyecto fue proporcionado por el NIH subvenciones P20RR015569, P20RR016460 y R01DA025755.

Este protocolo se ha modificado de Blair et al. 6. 1. LSD1 desmetilación Ensayo <ol><li> En un volumen final de 20 l, se combinan 125 LSD1 recombinante ng con 0,25 g de di-metilo histona H3 péptido (ARTKme2QTARKSTGGKAPRKQLYK-biotina) en tampón desmetilasa (50 mM Tris-Cl, pH 8,5, KCl 50 mM, 5 mM de MgCl 2, 5% glicerol). Además, realizar un control de péptido solo sin enzima. El control es de la sección 3 y no necesita la metilación reductiva. </li><li> Incubar durante 2 horas a 37 ° C. </li><li> Para recoger los péptidos, añadir 8 POROS l R2 20 micras perlas a cada muestra y agitar durante 15 minutos a temperatura ambiente. Preparar perlas POROS suspendiendo 1 volumen de perlas con un volumen de metanol, y luego añadir 10 volúmenes de 5% de ácido fórmico / 0,2% de TFA. </li><li> Condición dos C 18 ZipTips de aspiración 20 l de TFA al 0,1% en la punta y empujando hacia atrás con una pipeta. Hacer esto dos veces con 0,1% TFA, cuatro veces con 70% acetonitrilo / 0,1% TFA, y cuatro veces de nuevo con 0,1% de TFA. </li><li> Coloque las dos muestras (control y la reacción desmetilasa) en los condicionados C 18 ZipTips pipeteando la suspensión cordón detrás de la resina de C 18 en los ZipTips y empujando el volumen a través de una pipeta. </li><li> Lavar ZipTips dos veces con 20 l de TFA al 0,1%. </li><li> Eluir en 40 l de 70% acetonitrilo / 0,1% de TFA. </li><li> Liofilizar cada eluyente por completo con un concentrador SpeedVac. </li></ol> 2. La metilación reductiva Esta porción del protocolo es una modificación de Blair y cols. Y Rayment et al. 6,7. <ol><li> Vuelva a suspender la reacción desmetilasa en 100 l de tampón fosfato 50 mM, pH 7,4. </li><li> Para la reacción desmetilasa, añadir 8 l de 15 mg / ml de borano de dimetilamina. Borano dimetilamina se disolvió en tampón fosfato 50 mM, pH 7,4, para dar el 15 mg / ml de solución madre.Esto debe hacerse fresco. </li><li> Para la reacción desmetilasa, añadir 16 l de formaldehído 250 mM deuterado. El formaldehído 250 mM deuterado se prepara diluyendo el stock en H 2 0. Esto debe hacerse fresco. </li><li> Incubar a 4 ° C durante 2 horas. </li><li> Repita los pasos 2.2, 2.3 y 2.4. </li><li> Repita el paso 2.2. </li><li> Incubar a 4 ° C durante ~ 15 horas. </li><li> Para la muestra desmetilasa, añadir 12,5 l de Tris 1M, pH 7,4 para sofocar la reacción de metilación reductiva. </li></ol> 3. Espectrometría de masas MALDI <ol><li> Añadir POROS R2 20 micras cuentas a la reacción desmetilasa, y se acumulan en ZipTips como se describe en los pasos 1.3-1.6. </li></ol> Nota: Re-suspender el control en 100 l de tampón fosfato 50 mM, pH 7,4 y tratar como la otra muestra de partida en la etapa 3.1. <ol start="2"><li> Eluir el control y muestras desmetilasa de reacción de los ZipTips sobre una placa de muestra MALDI usando 2 l de 33% saturard ácido 2,5-dihidroxibenzoico. Para preparar el 33% de ácido 2,5-dihidroxibenzoico, hacer una solución saturada de ácido 2,5-dihidroxibenzoico en 70% de acetonitrilo / TFA al 0,1% y luego se diluye hasta 33% saturado en 70% de acetonitrilo / TFA al 0,1%. Permitir que las muestras eluidas a secar y cristalizar. </li><li> Recoge los espectros de masas con un PerkinElmerSciex MALDI-prOTOF espectrómetro de masas de alta resolución o disposición de espectrometría de masas MALDI 8,9,10. </li><li> Ver espectros y extraer áreas de los picos que utilizan el software PerkinElmerSciex TOFworks o software proporcionado por el fabricante del espectrómetro de masas. </li><li> Verifique que los productos de reacción por MS 2 con un sistema de espectrometría de masa disponible que proporciona capacidades de espectrometría de masas en tándem. </li></ol> 4. Análisis de Datos <ol><li> Con el fin de compensar el solapamiento isotópicos creados mediante el uso de formaldehído pesada (Figura 1B), determinar el área del pico (A) de 13 C 2 y 13 C relativ 4 isótopose para el pico monoisotópico para la muestra de control, que no ha sufrido metilación reductiva (Fig. 2A). La ecuación 1: 13C2 / A 12C = r 1 La ecuación 2: 13C4 / A 12C = r 2 Esto le dará la relación de péptido que existe en estos estados isotópica (r 1 y r 2) específica para su experimento y espectrómetro de masas. </li><li> Utilice la r 1 y los valores de r 2 en las siguientes fórmulas para cuantificar la cantidad relativa de péptido existente en cada estado modificación en la muestra de reacción desmetilasa (Fig. 2B): La ecuación 3: H3K4me2 = A-1 La ecuación 4: H3K4me = A 2 - R 1 (A 1) La ecuación 5: H3K4 = A 3 - r 2 (A 1) - [r 1 (A 2 - R 1 (A 1))] </li></ol> 5. Los resultados representativos Usando LSD1, se muestra la eficacia de MassSQUIRM para cuantificardesmetilación lisina. Como se describe en la sección de texto del protocolo, se realizó un ensayo desmetilasa con 125 ng de LSD1 y 0,25 g de di-metilo histona H3 péptido (ARTKme2QTARKSTGGKAPRKQLYK-biotina). El control y las muestras de desmetilasa reacción fueron sometidos a la metilación reductiva y espectrometría de masa MALDI. La reacción de control que no se sometieron a la metilación reductiva mostró la envoltura típica isotópica para este péptido y proporcionan áreas de los picos de las ecuaciones 1 y 2 (Figura 2A). El espectro de masas para la reacción desmetilasa proporcionan áreas de los picos de las ecuaciones 3-5 (Figura 2B). Uso de las áreas de los picos del control y la reacción desmetilasa, se determinó que el péptido LSD1 desmetilado para dar a los productos de reacción siguientes: el 33,7% di-metil Lys4, el 42,3% de mono-metil Lys4, y el 24% de la ONU-metilado Lys 4. Consulte a Blair et al. Para el análisis completo de la actividad LSD1 desmetilasa, así como inhibidores de 6 estudios. Nótese que las variables cambiantes, tales como, el tiempo de reacción de la enzimala concentración de la concentración y el sustrato que permitirá un análisis en profundidad de la actividad. <img alt="Figura 1" src="/files/ftp_upload/3604/3604fig1.jpg" /> Figura 1. Visión MassSQUIRM. (A) Un péptido N-terminal de la histona H3 se muestra como siendo sin-(verde), mono-(rojo), o di-(azul) metilado en lisina 4. La variación en la composición química de cada péptido conduce a la ionización diferencial haciendo complejo cuantificación. (B) metilación reductiva convierte todos los residuos de lisina en el estado de di-metilo que provoca que todos los péptidos para ionizar de manera similar. El uso de formaldehído pesado en la reacción de metilación reductiva permite la retención de la identidad de la metilación original. Los círculos vacíos indican metilación luz, mientras que los círculos cerrados indican metilación pesado. Modificado de Blair y cols. 2011 6. <img alt="Figura 2" src="/files/ftp_upload/3604/3604fig2.jpg" /> Figure 2. MassSQUIRM puede utilizarse para cuantificar péptidos diferencialmente metilado. (A) Un di-metilado sintético histona H3 péptido (ARTKme2QTARKSTGGKAPRKQLYK-biotina) se analizó mediante espectrometría de masas y los coeficientes máximos relativos al pico monoisotópico se observó como r 1 y r 2 en las ecuaciones 1 y 2. (B) El péptido sintético mismo se incubó con 125 ng LSD1 en tampón desmetilasa de dos horas a 37 ° C. Las muestras se sometieron entonces a MassSQUIRM análisis. Una población mixta de superposición de picos representa los tres estados diferentes de metilación como se ve en la Figura 1B. Las áreas bajo los picos monoisotopic se observa como A 1, A 2 y A 3. Áreas de los picos se utiliza en las ecuaciones 3-5. Los círculos vacíos indican metilación luz, mientras que los círculos cerrados indican metilación pesado. Modificado de Blair y cols. 2011 6.

<ol>
	<li>Goldberg, A. D., Allis, C. D., Bernstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+a+landscape+takes+shape.">Epigenetics: a landscape takes shape.</a> Cell. 128, 635-638 (2007).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Blair, L. P., Cao, J., Zou, M. R., Sayegh, J., Yan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+Regulation+by+Lysine+Demethylase+5+(KDM5)+Enzymes+in+Cancer.">Epigenetic Regulation by Lysine Demethylase 5 (KDM5) Enzymes in Cancer.</a> Cancers (Basel). 3, 1383-1404 (2011).</li><li>Cole, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+for+histone-modifying+enzymes.">Chemical probes for histone-modifying enzymes.</a> Nat. Chem. Biol. 4, 590-597 (2008).</li><li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylation+mediated+by+the+nuclear+amine+oxidase+homolog+LSD1.">Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.</a> Cell. 119, 941-953 (2004).</li><li>Blair, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MassSQUIRM:+An+assay+for+quantitative+measurement+of+lysine+demethylase+activity.">MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity.</a> Epigenetics. 6, 490-499 (2011).</li><li>Rayment, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+myosin+subfragment-1:+a+molecular+motor.">Three-dimensional structure of myosin subfragment-1: a molecular motor.</a> Science. 261, 50-508 (1993).</li><li>Collom, S. L., Jamakhandi, A. P., Tackett, A. J., Radominska-Pandya, A., Miller, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CYP2E1+active+site+residues+in+substrate+recognition+sequence+5+identified+by+photoaffinity+labeling+and+homology+modeling.">CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling.</a> Arch. Biochem. Biophys. 459, 59-69 (2007).</li><li>Gradolatto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+Yta7+Regulates+Histone+Gene+Expression.">Saccharomyces cerevisiae Yta7 Regulates Histone Gene Expression.</a> Genetics. 179, 291-304 (2008).</li><li>Taverna, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yng1+PHD+finger+binding+to+histone+H3+trimethylated+at+lysine+4+promotes+NuA3+HAT+activity+at+Lysine+14+of+H3+and+transcripiton+at+a+subset+of+targeted+ORFs.">Yng1 PHD finger binding to histone H3 trimethylated at lysine 4 promotes NuA3 HAT activity at Lysine 14 of H3 and transcripiton at a subset of targeted ORFs.</a> Mol. Cell. 24, 785-796 (2006).</li></ol>

No tenemos nada que revelar.

MassSQUIRM es un método económico y cuantitativo para el análisis integral de la actividad de la lisina desmetilasas involucrados en mono-y di-metilación. MassSQUIRM ofrece cuantificación no sólo del producto de la reacción, sino también para los productos intermedios. Este ensayo se puede utilizar como una poderosa herramienta en el estudio del mecanismo de desmetilasas histonas LSD1 y otros. También será útil para la clasificación de muchos recién descubiertos enzimas desmetilasa lisina como PHF8 y podrí...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> LSD1 </td><td> BPS Biociencias </td><td> 50100 </td></tr><tr><td> H3K4me2-biotina péptido </td><td> Elaboración propia </td><td> ninguno </td></tr><tr><td> Poros R2 20 micras cuentas </td><td> Applied Biosystems </td><td> 1-1129-06 </td></tr><tr><td> C 18 ZipTip </td><td> Millipore </td><td> ZTC18M </td></tr><tr><td> Ácido trifluoroacético (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> acetonitrilo </td><td> Pescador </td><td> A996 </td></tr><tr><td> 2,5-dihidroxibenzoico ácido </td><td> Sigma </td><td> 85707 </td></tr><tr><td> Borano de dimetilamina </td><td> Sigma </td><td> 180238 </td></tr><tr><td> isotópicamente pesada d 2-formaldehído </td><td> Cambridge Laboratorios de isótopos </td&gt; <td> DLM-805-20 </td></tr><tr><td> Tris </td><td> Pescador </td><td> BP154 </td></tr><tr><td> KCl </td><td> Pescador </td><td> BP366 </td></tr><tr><td> MgCl 2 </td><td> Pescador </td><td> BP214 </td></tr><tr><td> Glicerol </td><td> Pescador </td><td> G-33 </td></tr><tr><td> Ácido fórmico </td><td> Fluka </td><td> 06440 </td></tr><tr><td> El metanol </td><td> Pescador </td><td> A452 </td></tr><tr><td> Fosfato de Na </td><td> Pescador </td><td> BP329 </td></tr><tr><td> SpeedVac Concentrador </td><td> Sabio </td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF espectrómetro de masas y el software TOFworks </td><td> PerkinElmerSciex </td><td> ninguno </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

Recientemente, los reguladores epigenéticos se han descubierto como actores clave en muchas enfermedades diferentes 1-3. Como resultado, estas enzimas son los principales objetivos para los estudios de moléculas pequeñas y 4 desarrollo de fármacos. Muchos reguladores epigenéticos sólo recientemente se han descubierto y todavía están en proceso de ser clasificados. Entre estas enzimas son desmetilasas lisina que eliminan los grupos metilo de lisinas de las histonas y otras proteínas. Debido a la naturaleza novedosa de esta clase de enzimas, ensayos de pocos se han desarrollado para estudiar su actividad. Este ha sido un obstáculo en el camino tanto para la clasificación y el estudio de alto rendimiento de desmetilasas histonas. Actualmente, los ensayos desmetilasa existen muy pocos. Las que existen tienden a ser de naturaleza cualitativa y no se puede al mismo tiempo discernir entre los estados de metilación de lisina diferentes (un-, mono-, di-y tri-). La espectrometría de masas se utiliza comúnmente para determinar la actividad desmetilasa pero los ensayos actuales de espectrometría de masasno abordar si péptidos diferencialmente metilado ionizar diferente. Ionización diferencial de péptidos metilado hace comparando los estados de metilación difícil y ciertamente no cuantitativa (Figura 1A). Así, los ensayos disponibles no están optimizados para el análisis integral de la actividad desmetilasa. Aquí se describe un método llamado MassSQUIRM (cuantificación de espectrometría de masas utilizando metilación reductiva isotópica) que se basa en la metilación reductiva de grupos amina con formaldehído deuterado para forzar a todos lisinas ser di-metilado, lo que les hace esencialmente las mismas especies químicas y por lo tanto ionizar el misma (Figura 1B). La única diferencia química después de la metilación reductiva es hidrógeno y deuterio, que no afecta a la eficiencia de ionización MALDI. El ensayo MassSQUIRM es específico para los productos de reacción con desmetilasa lisinas un-, mono-o di-metilados. El ensayo es también aplicable a metiltransferasas lisina dando el saMe productos de reacción. Aquí, se utiliza una combinación de la química metilación reductiva y espectrometría de masa MALDI para medir la actividad de LSD1, un desmetilasa lisina capaz de eliminar di y mono-metil grupos, en un sustrato peptídico sintético 5. Este ensayo es simple y fácilmente susceptibles a cualquier laboratorio con el acceso a un espectrómetro de masas MALDI en el laboratorio oa través de una instalación de la proteómica. El ensayo tiene ~ 8 veces el rango dinámico y es fácilmente escalable para formato de placa 5.

Watch this Scientific Journal Video about Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity at JoVE.com

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Se presenta un método para el uso de espectrometría de masas MALDI y la química de la metilación reductiva para cuantificar los cambios en la metilación de la lisina.

Aplicación de MassSQUIRM para mediciones cuantitativas de la actividad desmetilasa Lisina

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

application-masssquirm-for-quantitative-measurements-lysine

Research

JoVE Journal

Biology

13.3K vistas.  University of Arkansas for Medical Sciences. Se presenta un método para el uso de espectrometría de masas MALDI y la química de la metilación reductiva para cuantificar los cambios en la metilación de la lisina.

Video: Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un ensayo para medir la actividad de Escherichia coli Inducible lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Investigación

Biología

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Cuantitativo, análisis en tiempo real de la actividad de reparación por escisión de base en lisados ​​celulares que utilizan la lesión específicos Molecular beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Análisis de la especificidad de la proteína lisina metiltransferasas Usando SPOT Péptido Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstitución funcional y la actividad del canal Las mediciones de Purificada Wildtype y mutante CFTR Proteína

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Desarrollo y caracterización de<em&gt; In Vitro</em&gt; Red de microvasos y mediciones cuantitativas de endotelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Y la producción de óxido nítrico

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

Aplicación de medición de electrofisiología para estudiar la actividad de transportadores de Electro-neutro

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Cuantificación de proteína específica lisina acetilación y Succinylation estequiometría usando Spectrometry total de la adquisición de datos-independiente

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.