Wir danken der UAMS Proteomics-Fazilität für die massenspektrometrische Unterstützung. Finanzierung für dieses Projekt wurde durch die NIH Zuschüsse P20RR015569, P20RR016460 und R01DA025755 zur Verfügung gestellt.

Dieses Protokoll wird von Blair et al modifiziert. 6. 1. LSD1 Demethylierung Assay <ol><li> In einem Endvolumen von 20 mL, 125 ng rekombinantem kombiniert mit 0,25 ug LSD1 Di-Methyl-Histon H3-Peptid (ARTKme2QTARKSTGGKAPRKQLYK-Biotin) im Demethylase-Puffer (50 mM Tris-Cl pH 8,5, 50 mM KCl, 5 mM MgCl 2, 5% Glycerin). Darüber hinaus führen Sie eine Peptid allein Kontrolle ohne Enzym. Die Steuerung ist für Abschnitt 3 und braucht keine reduktive Methylierung. </li><li> Inkubieren für 2 Stunden bei 37 ° C </li><li> Um die Peptide zu sammeln, fügen 8 ul POROS R2 20-Mikron-Perlen zu jeder Probe und agitieren für 15 Minuten bei Raumtemperatur. Bereiten POROS Perlen durch die Aussetzung ein Volumen von Perlen mit 1 Volumen Methanol, und fügen Sie dann 10 Volumen 5% Ameisensäure / 0,2% TFA. </li><li> Zustand zwei C 18 ZipTips durch Absaugen 20 uL 0,1% TFA in die Spitze und drücken Sie es wieder aus mit einer Pipette. Tun Sie dies zweimal mit 0,1% TFA, viermal mit 70% Acetonitril / 0,1% TFA, und viermal erneut mit 0,1% TFA. </li><li> Legen Sie die zwei Proben (Kontrolle und die Reaktion Demethylase) auf die konditionierte C 18 ZipTips durch Pipettieren die Kügelchenaufschlämmung hinter dem C 18-Harz in den ZipTips und schieben Sie die Lautstärke durch eine Pipette. </li><li> Waschen Sie ZipTips zweimal mit 20 ul 0,1% TFA. </li><li> Man eluiert in 40 ul 70% Acetonitril / 0,1% TFA. </li><li> Lyophilisieren jedes Elutionsmittel vollständig mit einer SpeedVac Konzentrator. </li></ol> 2. Reduktive Methylierung Dieser Teil des Protokolls wird von Blair et al modifiziert. Und Rayment et al. 6,7. <ol><li> Erneut zu suspendieren die Demethylase Reaktion in 100 ul 50 mM Phosphatpuffer, pH 7,4. </li><li> Um die Reaktion Demethylase, fügen 8 &amp;mgr; l 15 mg / ml Borandimethylamin. Boran-Dimethylamin in 50 mM Phosphatpuffer, pH 7,4 gelöst, die 15 mg / ml Stammlösung.Dies sollte frisch zubereitet werden. </li><li> Um die Reaktion Demethylase, fügen Sie 16 ul 250 mM deuteriertem Formaldehyd. Der 250 mm deuteriertem Formaldehyd wird durch Verdünnen der Lager in H 2 0 vorbereitet. Dies sollte frisch zubereitet werden. </li><li> Inkubieren bei 4 ° C für 2 Stunden. </li><li> Wiederholen Sie die Schritte 2.2, 2.3 &amp; 2.4. </li><li> Wiederholen Sie den Schritt 2.2. </li><li> Inkubieren bei 4 ° C für ca. 15 Stunden. </li><li> Zum Demethylase Probe wird mit 12,5 ul 1 M Tris, pH 7,4, um die reduktive Methylierung zu quenchen. </li></ol> 3. MALDI-Massenspektrometrie <ol><li> Fügen Sie POROS R2 20-Mikron-Perlen zum Demethylase Reaktion, und sammeln Sie auf ZipTips wie in den Schritten von 1,3 bis 1,6 beschrieben. </li></ol> Hinweis: Die Re-suspendieren die Kontrolle in 100 ul 50 mM Phosphatpuffer, pH 7,4 und zu behandeln, als die andere Probe ab Schritt 3.1. <ol start="2"><li> Eluieren die Kontrolle und Demethylase Reaktion Proben aus den ZipTips auf eine MALDI-Platte mit 2 ul von 33% zu sättigend 2,5-Dihydroxybenzoesäure. Zur Herstellung der 33% 2,5-Dihydroxybenzoesäure, machen Sie eine gesättigte Lösung von 2,5-Dihydroxybenzoesäure in 70% Acetonitril / 0,1% TFA verdünnt und dann auf 33% in 70% Acetonitril / 0,1% TFA gesättigt. Lassen Sie die eluierten Proben zu trocknen und kristallisieren. </li><li> Sammle Massenspektren mit einem PerkinElmerSciex MALDI-Massenspektrometer oder prOTOF verfügbar hochauflösende MALDI-Massenspektrometer 8,9,10. </li><li> Alle Spektren extrahieren und Peakflächen mit PerkinElmerSciex TOFworks Software oder Software, die vom Massenspektrometer Herstellers. </li><li> Stellen Sie sicher, Reaktionsprodukte von MS 2 mit einem verfügbaren massenspektrometrischen System, das Tandem-Massenspektrometrie-Fähigkeiten. </li></ol> 4. Datenanalyse <ol><li> Um den Isotopen Überlappung mit schweren Formaldehyd (1B) verursacht werden können, bestimmen die Peakfläche (A)-Verhältnis von 13 C 2 und 13 C 4-Isotopen relativee an den monoisotopischen Peak für die Kontrollprobe, die nicht reduktive Methylierung (2A) unterzogen wurde. Gl 1: Ein 13C2 / A 12C = r 1 Gl 2: Ein 13C4 / A 12C = r 2 Dadurch erhalten Sie das Verhältnis von Peptid bestehenden Zustände in diesen Isotopen (r 1 &amp; r 2) spezifisch für Ihr Experiment und Massenspektrometer. </li><li> Mit dem R 1 &amp; R 2-Werte in den folgenden Formeln zur Quantifizierung der relativen Menge an Peptid in den einzelnen Änderungen in der staatlichen Reaktion Demethylase Probe (Abb. 2B): Gl 3: H3K4me2 = A 1 Gleichung 4: H3K4me = A 2 - r 1 (A 1) Gleichung 5: H3K4 = A 3 - r 2 (A 1) - [r 1 (A 2 - r 1 (A 1))] </li></ol> 5. Repräsentative Ergebnisse Mit LSD1, zeigen wir die Wirksamkeit der MassSQUIRM zur QuantifizierungLysin-Demethylierung. Wie in dem Protokoll Text beschrieben, haben wir eine Demethylase-Assay mit 125 ng von LSD 1 und 0,25 ug Di-Methyl-Histon H3-Peptid (ARTKme2QTARKSTGGKAPRKQLYK-Biotin). Die Steuerung und Demethylase Reaktion Proben wurden reduktive Methylierung und MALDI Massenspektrometrie unterzogen. Die Kontroll-Reaktion, die nicht unterzogen wurden reduktive Methylierung zeigten die typische Isotopenverhältnis Umschlag für dieses Peptid und zur Verfügung gestellt Peakflächen für Gleichungen 1 und 2 (Abbildung 2A). Das Massenspektrum für die Reaktion zur Verfügung gestellt Demethylase Peakflächen für Gleichungen 3-5 (Abbildung 2B). Unter Verwendung der Peakflächen von der Steuerung und Demethylase Reaktion haben wir festgestellt, dass das Peptid LSD1 demethyliert, um die folgenden Reaktionsprodukte geben: 33,7% Di-Methyl Lys4, 42,3% mono-Methyl Lys4, und 24% un-methylierter Lys 4. Siehe Blair et al. Für die vollständige Analyse der LSD1 Demethylase sowie Inhibitorstudien 6. Beachten Sie, dass sich ändernden Variablen wie Reaktionszeit, EnzymKonzentrations-und Substrat-Konzentration wird für eine eingehende Analyse der Tätigkeit zu ermöglichen. <img alt="1" src="/files/ftp_upload/3604/3604fig1.jpg" /> Abbildung 1. MassSQUIRM Überblick. (A) ein N-terminales Peptid von Histon H3 wird dargestellt als un-(grün), Mono-(rot), oder Di-(blau) an Lysin 4 methyliert. Die Variation in der chemischen Zusammensetzung von jedem Peptid führt zu-Differential-Ionisation machen Quantifizierung komplex. (B) reduktive Methylierung konvertiert alle Lysinreste an den di-methyl Staat, der alle Peptide zu ionisieren, ähnlich verursacht. Die Verwendung von schweren Formaldehyd in der reduktiven Methylierung ermöglicht die Beibehaltung der Identität des ursprünglichen Methylierung. Offene Kreise zeigen Licht-Methylierung während geschlossene Kreise schweren Methylierung zeigen. Geändert von Blair et al. 2011 6. <img alt="2" src="/files/ftp_upload/3604/3604fig2.jpg" /> Figure 2. MassSQUIRM kann verwendet werden, um differentiell methylierte Peptide zu quantifizieren. (A)-A-di-methylierten synthetischen Histon H3-Peptid (ARTKme2QTARKSTGGKAPRKQLYK-Biotin) wurde unter Verwendung der Massenspektrometrie und Peak-Verhältnisse relativ zu dem monoisotopischen Peaks wurden als r 1 und r 2 in den Gleichungen 1 und 2 angegeben. (B) Das gleiche synthetische Peptid wurde mit 125 ng LSD1 in Demethylase Puffer für zwei Stunden bei 37 ° C inkubiert Die Proben wurden dann einer Analyse MassSQUIRM. Eine gemischte Bevölkerung von überlappenden Peaks stellt drei unterschiedliche Methylierung Zustände wie in 1B gesehen. Bereiche, die unter monoisotopischen Peaks wurden als A 1, A 2 und A 3 notiert haben. Die Peakflächen wurden in den Gleichungen 3-5 hatten. Offene Kreise zeigen Licht-Methylierung während geschlossene Kreise schweren Methylierung zeigen. Geändert von 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>

Wir haben nichts zu offenbaren.

MassSQUIRM ist eine preisgünstige und quantitative Methode zur umfassenden Analyse der Aktivität von Lysin Demethylasen in Mono-und Di-Methylierung beteiligt. MassSQUIRM bietet nicht nur die Quantifizierung des Produkts der Reaktion, sondern auch für die Zwischenprodukte. Dieser Test kann als ein mächtiges Werkzeug bei der Untersuchung des Mechanismus von LSD 1 und andere Histondemethylasen verwendet werden. Es sind auch nützlich für die Klassifizierung wie die neu entdeckten Lysin Demethylase Enzyme wie PHF8 und ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> LSD1 </td><td> BPS Biosciences </td><td> 50100 </td></tr><tr><td> H3K4me2-Biotin-Peptid </td><td> hergestellt im Haus </td><td> keiner </td></tr><tr><td> POROS R2 20-Mikron-Perlen </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> Trifluoressigsäure (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> Acetonitril </td><td> Fischer </td><td> A996 </td></tr><tr><td> 2,5-Dihydroxybenzoesäure </td><td> Sigma </td><td> 85707 </td></tr><tr><td> Borandimethylamin </td><td> Sigma </td><td> 180238 </td></tr><tr><td> isotopisch schwere d 2-Formaldehyd- </td><td> Cambridge Isotope Laboratories </td&gt; <td> DLM-805-20 </td></tr><tr><td> Tris </td><td> Fischer </td><td> BP154 </td></tr><tr><td> KCl </td><td> Fischer </td><td> BP366 </td></tr><tr><td> MgCl 2 </td><td> Fischer </td><td> BP214 </td></tr><tr><td> Glycerin </td><td> Fischer </td><td> G33 </td></tr><tr><td> Ameisensäure </td><td> Fluka </td><td> 06440 </td></tr><tr><td> Methanol </td><td> Fischer </td><td> A452 </td></tr><tr><td> Na-Phosphat </td><td> Fischer </td><td> BP329 </td></tr><tr><td> SpeedVac Concentrator </td><td> Gelehrter </td><td> DNA110 </td></tr><tr><td> MALDI-Massenspektrometer und prOTOF TOFworks Software </td><td> PerkinElmerSciex </td><td> keiner </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

In jüngster Zeit haben epigenetische Regulatoren als wichtige Akteure in vielen verschiedenen Krankheiten 3.1 entdeckt worden. Als ein Ergebnis sind diese Enzyme Hauptziele für kleine Moleküle Studien und Entwicklung von Arzneimitteln 4. Viele epigenetische Regulatoren haben erst vor kurzem entdeckt und sind noch im Gange klassifiziert. Unter diesen Enzymen sind Lysin Demethylasen die Methylgruppen von Lysine an Histonen und andere Proteine ​​zu entfernen. Durch die neuartige Charakter dieser Klasse von Enzymen, haben einige Tests entwickelt worden, um ihre Aktivität zu studieren. Dies war eine Straßensperre sowohl die Einstufung und hohem Durchsatz Studie Histondemethylasen. Derzeit existieren nur sehr wenige Demethylase Assays. Diejenigen, die es gibt, sind in der Regel in der Natur qualitativen und können nicht gleichzeitig zwischen den verschiedenen Lysin Methylierungszustände zu erkennen (un-, mono-, di und tri-). Die Massenspektrometrie wird häufig verwendet, um Demethylase-Aktivität zu bestimmen, aber die aktuellen massenspektrometrischen Assays tunnicht ansprechen, ob differentiell methylierte Peptide anders zu ionisieren. Differential-Ionisation von Peptiden methylierte macht den Vergleich Methylierung Staaten schwierig und schon gar nicht quantitativ (Abbildung 1A). So verfügbaren Assays sind nicht für die umfassende Analyse der Demethylase optimiert. Hier beschreiben wir ein Verfahren genannt MassSQUIRM (massenspektrometrische Quantifizierung unter Verwendung Isotopen reduktive Methylierung), die auf der reduktiven Methylierung Amingruppen mit deuteriertem Formaldehyd zu erzwingen, Lysine sein di-methylierten basiert, sodass sie im wesentlichen die gleichen chemischen Spezies und daher ionisieren gleichen (Abbildung 1B). Die einzige chemische Unterschied nach der reduktiven Methylierung ist Wasserstoff und Deuterium, die keinen Einfluss auf MALDI-Ionisierung Wirkungsgrade. Die MassSQUIRM Nachweis ist spezifisch für Demethylase Reaktionsprodukte mit un-, mono-oder di-methylierte Lysine. Der Assay ist auch anwendbar auf Lysin-Methyltransferasen geben die saIch Reaktionsprodukte. Wir verwenden hier eine Kombination aus der reduktiven Methylierung Chemie und MALDI-Massenspektrometrie, um die Aktivität von LSD 1 zu messen, eine Lysin-Demethylase entfernen kann di-und mono-Methylgruppen, einem synthetischen Peptid Substrat 5. Dieser Assay ist einfach und leicht zugänglich einem der Labor mit Zugang zu einem MALDI-Massenspektrometer im Labor oder durch eine Proteomik-Anlage. Der Test hat ~ 8-fach dynamischen Bereich und ist leicht skalierbar, um Platten-Format 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

Wir stellen eine Methode für die Verwendung von MALDI-Massenspektrometrie und reduktive Methylierung Chemie auf Veränderungen in Lysinmethylierung quantifizieren.

Die Anwendung des MassSQUIRM für quantitative Messungen von Lysin-Demethylase

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 Aufrufe.  University of Arkansas for Medical Sciences. Wir stellen eine Methode für die Verwendung von MALDI-Massenspektrometrie und reduktive Methylierung Chemie auf Veränderungen in Lysinmethylierung quantifizieren.

Serie: 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.

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin 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 ...

Forschung

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, Echtzeit-Analyse der Base Excision Repair Aktivität in Zelllysaten Verwendung läsionsspezifischen 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.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung in<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.

Cytosolischen Calcium Messungen in Nierenepithelzellen mittels Durchflusszytometrie

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.

Spezifitätsanalyse von Protein Lysin Methyltransferasen Mit SPOT Peptidarrays

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.

Funktionelle Rekonstitution und Kanalaktivitätsmessungen von gereinigtem Wildtyp und mutierten 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.

Entwicklung und Charakterisierung von<em&gt; In Vitro</em&gt; Mikrogefäßnetz und quantitative Messungen von Endothelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Und die Produktion von Stickoxid

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.

Anwendung der Elektrophysiologie Messung die Aktivität von Electro-neutralen Transporter zu studieren

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.

Quantifizierung der standortspezifischen Protein Lysin Acetylierung und Succinylation Stöchiometrie mittels Massenspektrometrie Daten-unabhängige Übernahme

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.