Agradecemos ao UAMS Facilidade Proteomics de apoio por espectrometria de massa. O financiamento para este projeto foi fornecido pelo NIH concede P20RR015569, P20RR016460 e R01DA025755.

Este protocolo é modificado a partir de Blair et al. 6. 1. LSD1 Ensaio desmetilação <ol><li> Em um volume final de 20 uL, combinar 125 ng LSD1 recombinante com 0,25 ug de di-metil histona H3 péptido (ARTKme2QTARKSTGGKAPRKQLYK-biotina) em tampão de desmetilase (50 mM Tris-Cl pH 8,5, 50 mM de KCl, 5 mM MgCl2, 5% glicerol). Além disso, realizar um péptido de controlo sozinha, sem enzima. O controle é para a secção 3 e não precisa de metilação redutiva. </li><li> Incubar durante 2 horas a 37 ° C. </li><li> Para recolher os péptidos, adicionar 8 uL Poros R2 20 micron grânulos para cada amostra e agitar durante 15 minutos à temperatura ambiente. Preparar grânulos Poros suspendendo 1 volume de esferas com um volume de metanol, e depois adicionar 10 volumes de 5% de ácido fórmico / TFA a 0,2%. </li><li> Condição dois C 18 ZipTips por aspiração de 20 uL de TFA 0,1% na ponta e empurrando-o de volta com uma pipeta. Faça isso duas vezes com T 0,1%FA, quatro vezes com acetonitrilo 70% / TFA a 0,1%, e quatro vezes novamente com TFA a 0,1%. </li><li> Carregar as duas amostras (controle ea reacção desmetilase) sobre os condicionado C 18 ZipTips pipetando a lama grânulo por trás da resina C 18 nas ZipTips e empurrando o volume através com um pipetador. </li><li> Lavar duas vezes com ZipTips 20 uL de TFA a 0,1%. </li><li> Eluir em 40 uL de acetonitrilo 70% / TFA a 0,1%. </li><li> Liofilizar cada eluente completamente com um concentrador Speedvac. </li></ol> 2. Metilação redutiva Esta porção do protocolo é modificado a partir de Blair et al. E Rayment et al. 6,7. <ol><li> Re-suspender a reacção desmetilase em 100 uL de tampão fosfato 50 mM, pH 7,4. </li><li> Para a reacção de desmetilase, adicionar 8 uL de 15 mg / mL de borano dimetilamina. Borano dimetilamina é dissolvido em tampão fosfato 50 mM, pH 7,4, para dar o 15 mg / ml de solução concentrada.Isto deve ser feito fresco. </li><li> Para a reacção de desmetilase, adicionar 16 uL de 250 mM de formaldeído deuterado. O formaldeído 250 mM deuterado é preparada diluindo o estoque em H 2 0. Isto deve ser feito fresco. </li><li> Incubar a 4 ° C durante 2 horas. </li><li> Repita os passos 2.2, 2.3 e 2.4. </li><li> Repita o passo 2.2. </li><li> Incubar a 4 ° C durante ~ 15 horas. </li><li> Para a amostra desmetilase, adicionar 12,5 ul de 1M Tris, pH 7,4 para extinguir a reacção de metilação redutiva. </li></ol> 3. Espectrometria de Massa MALDI <ol><li> Adicionar Poros R2 20 micron grânulos para a reacção desmetilase, e recolher em ZipTips como descrito nos passos 1,3-1,6. </li></ol> Nota: Re-suspende o controle em 100 uL de tampão fosfato 50 mM, pH 7,4 e tratar como a outra amostra de partida no passo 3.1. <ol start="2"><li> Eluir o controlo e as amostras reaccionais desmetilase a partir dos ZipTips sobre uma placa de amostra MALDI usando 2 uL de 33% saturard ácido 2,5-dihidroxibenzóico. Para preparar a 33% de ácido 2,5-dihidroxibenzoico, fazer uma solução saturada de 2,5-di-hidroxibenzóico em acetonitrilo 70% / TFA a 0,1% e, em seguida, diluir a 33% saturado em acetonitrilo 70% / TFA a 0,1%. Permitir que as amostras eluidas para secar e cristalizar. </li><li> Coletar espectros de massa usando um espectrômetro de massa MALDI PerkinElmerSciex-prOTOF ou disponível de alta resolução espectrômetro de massa MALDI 8,9,10. </li><li> Ver espectros e extrair áreas dos picos usando PerkinElmerSciex software TOFworks ou software fornecido pelo fabricante do espectrômetro de massa. </li><li> Verificar produtos de reacção por MS 2, com um sistema de massa disponível por espectrometria de proporcionar capacidades de espectrometria de massa em tandem. </li></ol> 4. Análise de Dados <ol><li> A fim de compensar a sobreposição isotópicos criadas usando formaldeído pesada (Figura 1B), determinar a área do pico (A) proporção de 13 C e 13 C 2 4 isótopos relative para o pico monoisotópico para a amostra de controle, que não foi objecto de metilação redutiva (Fig. 2A). Eq. 1: Um 13C2 / A 12C = r 1 Eq. 2: Uma 13C4 / A 12C = r 2 Isto lhe dará a relação do peptídeo existentes nesses estados isotópica (r 1 e r 2) específico para o seu experimento e espectrômetro de massa. </li><li> Utilizar a r 1 e valores de r 2 nas fórmulas seguintes para quantificar a quantidade relativa de péptido existentes em cada estado modificação na amostra reacção desmetilase (Fig. 2B): Eq. 3: H3K4me2 = A 1 Eq. 4: H3K4me = A 2 - r 1 (A 1) Eq. 5: H3K4 = A 3 - r 2 (A 1) - [r 1 (A 2 - r 1 (A 1))] </li></ol> 5. Os resultados representativos Usando LSD1, mostramos a eficácia do MassSQUIRM para quantificardesmetilação lisina. Como descrito na secção de Texto protocolo, realizou-se um ensaio de desmetilase com 125 ng de LSD1 e 0,25 ug de di-metil histona H3 péptido (ARTKme2QTARKSTGGKAPRKQLYK-biotina). O controlo e as amostras reaccionais desmetilase foram submetidos a metilação redutiva e espectrometria de massa MALDI. A reacção de controlo que não foi submetido a metilação redutiva mostrou o envelope típico isotópica para este péptido, e desde as áreas dos picos para as equações 1 e 2 (Figura 2A). O espectro de massa para a reacção de desmetilase desde áreas de pico para equações 3-5 (Figura 2B). Usando as áreas dos picos do controlo e reacção desmetilase, determinou-se que o péptido LSD1 desmetilado para dar os produtos de reacção seguintes: 33,7% de di-metil Lys4, 42,3% de mono-metil Lys4, e 24% un-metilado Lys 4. Consulte a Blair et al. Para a análise completa de LSD1 atividade demetilase bem como estudos de inibidores 6. Note-se que as variáveis ​​mutação tais como o tempo de reacção da enzima,concentração de concentração e substrato permitirá uma análise aprofundada da atividade. <img alt="A Figura 1" src="/files/ftp_upload/3604/3604fig1.jpg" /> Figura 1. Resumo MassSQUIRM. (A) um péptido N-terminal a partir de histona H3 é mostrado como sendo un-(verde), mono-(vermelho), ou di-(azul) metilado na lisina 4. A variação na composição química de cada péptido leva a ionização diferencial tornando complexo quantificação. (B) metilação redutiva converte todos os resíduos de lisina para o estado de di-metil que faz com que todos os péptidos para ionizar de forma semelhante. A utilização de formaldeído pesado na reacção de metilação redutiva permite a retenção da identidade do metilação original. Círculos abertos indicam metilação luz, enquanto círculos fechados indicam metilação pesado. Modificado de Blair et al. 2011 6. <img alt="A Figura 2" src="/files/ftp_upload/3604/3604fig2.jpg" /> Figur2 e. MassSQUIRM pode ser usado para quantificar os péptidos diferencialmente metilados. (A) Um di-metilado sintético histona H3 péptido (ARTKme2QTARKSTGGKAPRKQLYK-biotina) foi analisado por espectrometria de massa e os rácios dos picos relativos ao pico monoisotópico foram anotados como R1 e R 2 nas equações 1 e 2. (B) O péptido sintético mesmo foi incubado com 125 ng LSD1, em tampão de desmetilase durante duas horas a 37 ° C. As amostras foram então submetidas a análise MassSQUIRM. Uma população mista de sobreposição picos representa três estados diferentes de metilação tal como são vistos na Figura 1B. As áreas sob os picos monoisotópico foram notadas como um 1, A 2, e A 3. Áreas de pico foram utilizados nas equações 3-5. Círculos abertos indicam metilação luz, enquanto círculos fechados indicam metilação pesado. Modificado de Blair et al. 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>

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MassSQUIRM é um método barato e quantitativa para análise abrangente da atividade de demethylases lisina envolvidas na mono-e di-metilação. MassSQUIRM oferece não só a quantificação do produto da reacção, mas também para os intermediários. Este ensaio pode ser utilizado como uma ferramenta poderosa para estudar o mecanismo de LSD1 e outros demethylases histona. Também irá ser útil para a classificação de muitas enzimas recém-descobertas desmetilase de lisina, tais como PHF8 e poderiam ser usados ​?...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> LSD1 </td><td> BPS Biosciences </td><td> 50100 </td></tr><tr><td> H3K4me2-biotina péptido </td><td> preparada em casa </td><td> nenhum </td></tr><tr><td> POROS R2 20 esferas de micro </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> acetonitrila </td><td> Pescador </td><td> A996 </td></tr><tr><td> 2,5-di-hidroxibenzóico </td><td> Sigma </td><td> 85707 </td></tr><tr><td> Borano dimetilamina </td><td> Sigma </td><td> 180238 </td></tr><tr><td> isotopicamente pesado d 2-formaldeído </td><td> Cambridge Isotope Laboratories </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> Glicerina </td><td> Pescador </td><td> G33 </td></tr><tr><td> O ácido fórmico </td><td> Fluka </td><td> 06440 </td></tr><tr><td> Metanol </td><td> Pescador </td><td> A452 </td></tr><tr><td> Na-fosfato </td><td> Pescador </td><td> BP329 </td></tr><tr><td> Concentrador Speedvac </td><td> Sábio </td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF espectrômetro de massa e TOFworks software </td><td> PerkinElmerSciex </td><td> nenhum </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

Recentemente, os reguladores epigenéticos têm sido descobertos como atores-chave em muitos diferentes doenças 1-3. Como resultado, estas enzimas são alvos principais para os estudos de moléculas pequenas e 4 desenvolvimento de drogas. Muitos reguladores epigenéticos têm apenas recentemente descobertos e ainda estão em vias de ser classificado. Entre estas enzimas são demethylases lisina que removem grupos metilo a partir de lisinas na histonas e outras proteínas. Devido à natureza novo desta classe de enzimas, ensaios de poucos têm sido desenvolvidos para estudar a sua actividade. Este tem sido um bloqueio na estrada para tanto a classificação e estudo de alto rendimento de demethylases histonas. Atualmente, ensaios demetilase existem poucos. As que existem tendem a ser de natureza qualitativa e não pode discernir entre simultaneamente os diferentes estados de metilação de lisina (un-, mono-, di-e tri-). Espectrometria de massa é comumente usado para determinar a atividade demetilase mas atuais ensaios de espectrometria de massa donão dirigir se peptídeos diferencialmente metilados ionizar de forma diferente. Ionização diferencial de péptidos metilados faz comparando estados de metilação difícil e certamente não quantitativa (Figura 1A). Assim, os ensaios disponíveis não são otimizados para a análise global da atividade demetilase. Descrevemos aqui um método chamado MassSQUIRM (quantificação por espectrometria de massa utilizando metilação redutiva isotópica) que é baseado em metilação redutiva de grupos amina com formaldeído deuterado para forçar todos os lisinas ser di-metilado, tornando-as assim essencialmente os mesmos espécies químicas e, portanto, ionizar o mesmo (Figura 1B). A diferença química apenas após a metilação redutiva é hidrogénio e deutério, que não afecta a eficiência de ionização MALDI. O ensaio MassSQUIRM é específico para produtos de reacção desmetilase com lisinas un-, mono-ou di-metilados. O ensaio é também aplicável a metiltransferases lisina dando o same produtos de reacção. Aqui, nós usar uma combinação de química metilação redutiva e espectrometria de massa MALDI para medir a actividade de LSD1, um desmetilase lisina capaz de remover di-e mono-metilo grupos, sobre um substrato de péptido sintético 5. Este ensaio é simples e facilmente acessíveis a qualquer laboratório com acesso a um espectrómetro de massa MALDI em laboratório ou através de um mecanismo proteômica. O ensaio tem ~ gama de 8 vezes dinâmico e é prontamente adaptar ao 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

Apresentamos um método para o uso de espectrometria de massa MALDI e química metilação redutiva para quantificar as mudanças na metilação da lisina.

Aplicação de Medidas para MassSQUIRM quantitativa da actividade demetilase 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

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Biology

13.3K visualizações.  University of Arkansas for Medical Sciences. Apresentamos um método para o uso de espectrometria de massa MALDI e química metilação redutiva para quantificar as mudanças na metilação da lisina.

Vídeo: 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.

Um ensaio para medir a atividade do Escherichia coli Induzível 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 ...

Biologia

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.

Quantitativa, análise em tempo real da Base de Dados de atividade de reparo de excisão em lisados ​​celulares Utilizando Lesão específicos Beacons Molecular

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.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<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.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

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.

Especificidade Análise de Proteína Lisina Metiltransferases Usando 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 ...

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.

Reconstituição funcional e atividade do canal Medidas de purificada tipo selvagem e mutante CFTR Protein

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.

Desenvolvimento e Caracterização de<em&gt; In Vitro</em&gt; Rede de microvasos e medições quantitativas de endotelial [ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E óxido nítrico Produção

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.

Aplicação de medida de eletrofisiologia para estudar a atividade 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.

Quantificação de Estoicometria de Acetilação e Succinilação de Lisinas Proteicas em Sítios Específicos Usando Espectrometria de Massa de Aquisição de Dados Independente

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.