Nous tenons à remercier le Fonds pour l&#39;UAMS protéomique pour le soutien par spectrométrie de masse. Le financement de ce projet a été fourni par des subventions du NIH P20RR015569, P20RR016460 et R01DA025755.

Ce protocole est modifié de Blair et al. 6. 1. LSD1 Assay déméthylation <ol><li> Dans un volume final de 20 ul, mélanger 125 ng LSD1 recombinant avec 0,25 pg di-méthyl histone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotine) dans un tampon déméthylase (50 mM Tris-Cl pH 8,5, KCl 50 mM, 5 mM MgCl 2, 5% glycérol). En outre, d&#39;effectuer un contrôle peptide seul, sans enzyme. Le contrôle est de la section 3 et n&#39;a pas besoin de méthylation réductrice. </li><li> Incuber pendant 2 heures à 37 ° C. </li><li> Pour recueillir les peptides, ajouter 8 ul POROS perles R2 microns de 20 à chaque échantillon et agiter pendant 15 minutes à température ambiante. Préparer perles POROS en suspendant 1 volume de perles avec 1 volume de méthanol, puis ajouter 10 volumes d&#39;acide formique à 5% / TFA 0,2%. </li><li> Condition deux C 18 ZipTips par aspiration 20 pl de TFA 0,1% dans la pointe et le repoussant avec une pipette. Pour ce faire, deux fois avec T 0,1%FA, quatre fois avec de l&#39;acétonitrile 70% / TFA 0,1%, et quatre fois encore avec 0,1% de TFA. </li><li> Chargez les deux échantillons (contrôle et la réaction déméthylase) sur les C 18 climatisées ZipTips par pipetage la bouillie bourrelet derrière la résine C 18 dans les ZipTips et en poussant le volume à travers avec une pipette. </li><li> Laver deux fois avec 20 ZipTips ul de 0,1% de TFA. </li><li> Eluer dans 40 uL d&#39;acétonitrile 70% / 0,1% de TFA. </li><li> Lyophiliser chaque éluant complètement avec un concentrateur SpeedVac. </li></ol> 2. Méthylation réductrice Cette partie du protocole est modifié de Blair et al. Et Rayment et al. 6,7. <ol><li> Re-suspendre la réaction déméthylase dans 100 ul de tampon phosphate 50 mM, pH 7,4. </li><li> Pour la réaction déméthylase, ajouter 8 pi de 15 mg / ml de borane diméthylamine. Borane diméthylamine est dissous dans un tampon phosphate 50 mM, pH 7,4, pour donner 15 mg / ml solution stock.Cela devrait être fait frais. </li><li> Pour la réaction déméthylase, ajouter 16 ul de 250 mM de formaldéhyde deutéré. Les 250 mM de formaldéhyde deutéré est préparé en diluant le stock en H 2 0. Cela devrait être fait frais. </li><li> Incuber à 4 ° C pendant 2 heures. </li><li> Répétez les étapes 2.2, 2.3 et 2.4. </li><li> Répétez l&#39;étape 2.2. </li><li> Incuber à 4 ° C pendant environ 15 heures. </li><li> Pour l&#39;échantillon déméthylase, ajouter 12,5 ul de Tris 1M, pH 7,4 pour étancher la réaction de méthylation réductrice. </li></ol> 3. Spectrométrie de masse MALDI <ol><li> Ajouter POROS perles R2 microns 20 à la réaction déméthylase, et de recueillir sur ZipTips tel que décrit dans les étapes 1.3-1.6. </li></ol> Remarque: Re-suspendre le contrôle dans 100 ul de tampon phosphate 50 mM, pH 7,4 et traiter comme l&#39;autre échantillon de départ à l&#39;étape 3.1. <ol start="2"><li> Éluer le contrôle et les échantillons de réaction déméthylase des ZipTips sur une plaque échantillon MALDI l&#39;aide de 2 ul de 33% de saturerd acide 2,5-dihydroxybenzoïque. Pour préparer les 33% d&#39;acide 2,5-dihydroxybenzoïque, préparer une solution saturée de 2,5-dihydroxybenzoïque dans l&#39;acétonitrile 70% / 0,1% de TFA, puis diluer à 33% de graisses saturées dans l&#39;acétonitrile 70% / 0,1% de TFA. Laisser les échantillons élués à sécher et à se cristalliser. </li><li> Recueillir des spectres de masse en utilisant un spectromètre de masse MALDI-PerkinElmerSciex prOTOF ou disponibles en haute résolution de masse MALDI spectromètre de 8,9,10. </li><li> Voir spectres et d&#39;extraire les zones de pointe utilisant PerkinElmerSciex TOFworks logiciel ou logiciel fourni par le fabricant spectromètre de masse. </li><li> Vérifiez les produits de réaction par la SP 2 avec un système de masse disponible par spectrométrie de masse en tandem offrant des capacités de spectrométrie. </li></ol> 4. Analyse des données <ol><li> Afin de compenser pour le chevauchement des isotopes créés à l&#39;aide de formaldéhyde lourde (figure 1B), de déterminer l&#39;aire du pic (A) ratio de 13 C 2 et C 13 relativ 4 isotopese au pic monoisotopique pour l&#39;échantillon de contrôle, qui n&#39;a pas subi de méthylation réductrice (fig. 2A). Eq 1: Un 13C2 / A 12C = r 1 Eq 2: Un 13C4 / A 12C = r 2 Cela vous donnera le rapport de peptide existant dans ces États isotopique (r 1 &amp; r 2) spécifique à votre expérience et spectromètre de masse. </li><li> Utilisez le r 1 &amp; r 2 valeurs dans les formules suivantes pour mesurer la quantité relative de peptide existant dans chaque état ​​de modification de l&#39;échantillon de réaction déméthylase (Fig. 2B): Eq 3: H3K4me2 = A 1 Eq 4: H3K4me = A 2 - r 1 (A 1) Eq 5: H3K4 = A 3 - r 2 (1 A) - [r 1 (A 2 - r 1 (A 1))] </li></ol> 5. Les résultats représentatifs Utilisation LSD1, nous montrons l&#39;efficacité de MassSQUIRM pour quantifierdéméthylation lysine. Comme décrit dans la section Texte protocole, nous avons effectué un test avec 125 ng déméthylase de LSD1 et 0,25 pg di-méthyl histone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotine). Le contrôle et des échantillons de réaction déméthylase ont été soumis à une méthylation réductrice et spectrométrie de masse MALDI. La réaction de contrôle qui n&#39;a pas subi de méthylation réductrice a montré l&#39;enveloppe typique isotopique pour ce peptide et à condition domaines de pointe pour les équations 1 et 2 (figure 2A). Le spectre de masse pour la réaction déméthylase fourni domaines de pointe pour les équations 3-5 (figure 2B). Utilisation des aires des pics de la commande et la réaction déméthylase, on a déterminé que le peptide LSD1 déméthylé pour donner les produits de réaction suivants: 33,7% de di-méthyl lysine 4, 42,3% de mono-méthyl lysine 4, et 24% non méthylé Lys 4. Reportez-vous à Blair et al. Pour l&#39;analyse complète de l&#39;activité déméthylase LSD1 ainsi que 6 études inhibiteurs. Notez que les variables telles que l&#39;évolution des temps de réaction, une enzymela concentration de concentration et le substrat sera de permettre une analyse en profondeur de l&#39;activité. <img alt="Figure 1" src="/files/ftp_upload/3604/3604fig1.jpg" /> Figure 1. Aperçu MassSQUIRM. (A) un peptide N-terminal de l&#39;histone H3 est représentée comme étant non-(vert), mono-(rouge), ou di-(bleu) méthylé en lysine 4. Variation de la composition chimique de chaque peptide conduit à l&#39;ionisation différentielle rendant complexe la quantification. (B) méthylation réductrice convertit tous les résidus lysine à l&#39;état di-méthyl qui provoque tous les peptides pour ioniser la même façon. L&#39;utilisation de formaldéhyde lourde dans la réaction de méthylation réductrice permet la rétention de l&#39;identité de la méthylation d&#39;origine. Les cercles ouverts indiquent la méthylation de lumière tandis que les cercles fermés indiquent méthylation lourde. Modification de Blair et al. 2011 6. <img alt="Figure 2" src="/files/ftp_upload/3604/3604fig2.jpg" /> Figure 2. MassSQUIRM peut être utilisée pour quantifier de façon différentielle peptides méthylés. (A) une di-méthylé synthétique histone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotine) est analysé par spectrométrie de masse et le ratio de pointe par rapport à la crête monoisotopique ont été noté que r 1 et r 2 dans les équations 1 et 2. (B) Le peptide synthétique même a été incubé avec LSD1 ng 125 dans un tampon de déméthylase pour deux heures à 37 ° C. Les échantillons ont été ensuite soumis à l&#39;analyse MassSQUIRM. Une population mixte de chevauchement des pics représente trois états de méthylation différentes comme on le voit dans la figure 1B. Domaines relevant des pics mono isotopiques ont été notés comme A 1, A 2 et A 3. Domaines de pointe ont été utilisées dans les équations 3-5. Les cercles ouverts indiquent la méthylation de lumière tandis que les cercles fermés indiquent méthylation lourde. Modification 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>

Nous n&#39;avons rien à communiquer.

MassSQUIRM est une méthode peu coûteuse et quantitative pour l&#39;analyse détaillée de l&#39;activité de déméthylases lysine impliqués dans les mono-et di-méthylation. MassSQUIRM offre quantification non seulement du produit de la réaction, mais aussi pour les produits intermédiaires. Ce test peut être utilisé comme un outil puissant pour étudier le mécanisme de LSD1 et autres déméthylases histones. Il sera également utile pour classer les nombreuses enzymes nouvellement découverts déméthylase lysi...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> LSD1 </td><td> BPS Biosciences </td><td> 50100 </td></tr><tr><td> H3K4me2-biotine peptide </td><td> préparés à l&#39;interne </td><td> aucun </td></tr><tr><td> POROS R2 20 perles micron </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> L&#39;acide trifluoroacétique (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> acétonitrile </td><td> Pêcheur </td><td> A996 </td></tr><tr><td> Acide 2,5-dihydroxybenzoïque </td><td> Sigma </td><td> 85707 </td></tr><tr><td> Borane diméthylamine </td><td> Sigma </td><td> 180238 </td></tr><tr><td> isotopiquement lourd d 2-formaldéhyde </td><td> Cambridge Isotope Laboratories </td&gt; <td> DLM-805-20 </td></tr><tr><td> Tris </td><td> Pêcheur </td><td> BP154 </td></tr><tr><td> KCl </td><td> Pêcheur </td><td> BP366 </td></tr><tr><td> MgCl 2 </td><td> Pêcheur </td><td> BP214 </td></tr><tr><td> Glycérol </td><td> Pêcheur </td><td> G33 </td></tr><tr><td> L&#39;acide formique </td><td> Fluka </td><td> 06440 </td></tr><tr><td> Le méthanol </td><td> Pêcheur </td><td> A452 </td></tr><tr><td> Phosphate de Na </td><td> Pêcheur </td><td> BP329 </td></tr><tr><td> Concentrateur SpeedVac </td><td> Savant </td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF spectromètre de masse et TOFworks logiciels </td><td> PerkinElmerSciex </td><td> aucun </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

Récemment, les régulateurs épigénétiques ont été découverts comme des acteurs clés dans de nombreuses maladies différentes 1-3. En conséquence, ces enzymes sont des cibles de choix pour les études de petites molécules et 4 développement des médicaments. De nombreux régulateurs épigénétiques n&#39;ont été que récemment découvert et sont encore en train d&#39;être classés. Parmi ces enzymes sont déméthylases lysine qui éliminent des groupes méthyle de lysines sur les histones et autres protéines. En raison de la nature de cette nouvelle classe d&#39;enzymes, des dosages peu ont été développés pour étudier leur activité. Cela a été un barrage routier à la fois la classification et l&#39;étude à haut débit de déméthylases histones. Actuellement, des essais déméthylase il existe très peu. Celles qui existent ont tendance à être de nature qualitative et ne peut pas simultanément discerner entre les différents états de méthylation de lysine (non, mono-, di-et tri-). La spectrométrie de masse est couramment utilisé pour déterminer l&#39;activité déméthylase, mais de masse actuels dosages par spectrométrie de fairepas non plus si les peptides différentiellement méthylés ioniser différemment. Ionisation différentielle de peptides méthylés permet de comparer les états de méthylation difficile et certainement pas quantitative (figure 1A). Ainsi des essais disponibles ne sont pas optimisés pour l&#39;analyse complète de l&#39;activité déméthylase. Nous décrivons ici une méthode appelée MassSQUIRM (masse quantification par spectrométrie isotopique utilisant méthylation réductrice) qui est basé sur la méthylation réductrice de groupes amine avec le formaldéhyde deutéré pour forcer tous les lysines être di-méthylé, ce qui les rend essentiellement les mêmes espèces chimiques et donc ioniser l&#39; même (figure 1B). La différence chimique seulement après la méthylation réductrice est de l&#39;hydrogène et de deutérium, qui n&#39;affecte pas l&#39;efficacité d&#39;ionisation MALDI. Le dosage MassSQUIRM est spécifique pour les produits de réaction avec déméthylase lysines non, mono-ou di-méthylés. Le dosage est également applicable aux méthyltransférases lysine donnant la SAmoi produits de réaction. Ici, nous utilisons une combinaison de la chimie méthylation réductrice et spectrométrie de masse MALDI pour mesurer l&#39;activité de LSD1, une déméthylase lysine capable d&#39;éliminer di-et mono-méthyle, sur un substrat peptide synthétique 5. Ce test est simple et facile de les faire n&#39;importe quel laboratoire d&#39;avoir accès à un spectromètre de masse MALDI dans le laboratoire ou par l&#39;intermédiaire d&#39;une installation protéomique. L&#39;essai a ~ 8 fois la plage dynamique et est facilement extensible à 5 format de plaque.

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

Nous présentons une méthode pour l&#39;utilisation de la spectrométrie de masse MALDI et de la chimie méthylation réductrice pour quantifier les changements dans la méthylation de la lysine.

Application de la MassSQUIRM pour des mesures quantitatives de l&#39;activité Déméthylase Lysine

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 ...

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13.3K vues.  University of Arkansas for Medical Sciences. Nous présentons une méthode pour l'utilisation de la spectrométrie de masse MALDI et de la chimie méthylation réductrice pour quantifier les changements dans la méthylation de la lysine.

Vidéo: 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 test pour mesurer l&#39;activité des Escherichia coli Inductible 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 ...

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Biologie

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.

Quantitative, en temps réel Analyse de l&#39;activité de réparation par excision de base dans les lysats cellulaires Utilisant des lésions spécifiques Beacons moléculaires

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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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.

Mesures de calcium cytosolique dans les cellules épithéliales rénales par cytométrie en flux

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.

Spécificité Analyse des protéines lysine Methyltransferases Utilisation 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.

Reconstitution fonctionnelle et Activité du Chan mesures de type sauvage purifiée et Mutant protéine CFTR

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.

Développement et caractérisation de<em&gt; In vitro</em&gt; Microvaisseaux réseau et des mesures quantitatives de l&#39;endothélium [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Et production d&#39;oxyde nitrique

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.

Application de mesure de l’électrophysiologie pour étudier l’activité des transporteurs de l’Electro-neutre

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.

Quantification des protéines spécifiques au site Lysine acétylation et stoechiométrie Succinylation spectrométrie de masse indépendante des données Acquisition

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.