Name: application of masssquirm for quantitative measurements of lysine demethylase activity
Uploaded: 2012-03-11T14:00:00
Description: Watch this Scientific Journal Video about Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity at JoVE.com

Vi takker UAMS Proteomics Facility for massespektrometrisk støtte. Finansieringen af ​​dette projekt blev leveret af NIH tilskud P20RR015569, P20RR016460 og R01DA025755.

Denne protokol er modificeret fra Blair et al. 6. 1. LSD1 demetylering Assay <ol><li> I et slutvolumen på 20 ul, kombinere 125 ng rekombinant LSD1 med 0,25 ug di-methyl histon H3 peptid (ARTKme2QTARKSTGGKAPRKQLYK-biotin) i demethylase puffer (50 mM Tris-Cl pH 8,5, 50 mM KCI, 5 mM MgCl2, 5% glycerol). Desuden udføres en peptid alene kontrol uden enzym. Styringen er for afsnit 3 og behøver ikke reduktiv methylering. </li><li> Inkuber i 2 timer ved 37 ° C. <...

<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>

MassSQUIRM er en billig og kvantitativ metode til omfattende analyse af aktiviteten af ​​lysin demethylases involveret i mono-og di-methylering. MassSQUIRM giver kvantificering ikke blot af produktet af reaktionen, men også for mellemprodukterne. Denne analyse kan bruges som et effektivt redskab i at studere mekanismen for LSD1 og andre histon demethylases. Det vil også være nyttige til klassificering mange nyopdagede lysin demethylase enzymer, såsom PHF8 og kan anvendes til visse methyltransferase enzymer.

<table border="1"><tbody><tr><td> Navn på reagenset </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> fremstillet in-house </td><td> Ingen </td></tr><tr><td> POROS R2 20 mikron perler </td><td> Applied Biosystems </td><td> 1-1129-06 </td></tr><tr><td> C18 ZipTip </td><td> Millipore </td><td> ZTC18M </td></tr><tr><td> Trifluoreddikesyre (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> acetonitril </td><td> Fisher </td><td> A996 </td></tr><tr><td> 2,5-dihydroxybenzoesyre </td><td> Sigma </td><td> 85707 </td></tr><tr><td> Boran dimethylamin </td><td> Sigma </td><td> 180238 </td></tr><tr><td> isotopisk tunge d-2-formaldehyd </td><td> Cambridge Isotope Laboratories </td&gt; <td> DLM-805-20 </td></tr><tr><td> Tris </td><td> Fisher </td><td> BP154 </td></tr><tr><td> KCl </td><td> Fisher </td><td> BP366 </td></tr><tr><td> MgCl2 </td><td> Fisher </td><td> BP214 </td></tr><tr><td> Glycerol </td><td> Fisher </td><td> G33 </td></tr><tr><td> Myresyre </td><td> Fluka </td><td> 06440 </td></tr><tr><td> Methanol </td><td> Fisher </td><td> A452 </td></tr><tr><td> Na-phosphat </td><td> Fisher </td><td> BP329 </td></tr><tr><td> SpeedVac Concentrator </td><td> Savant </td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF massespektrometer og TOFworks software </td><td> PerkinElmerSciex </td><td> Ingen </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

For nylig har epigenetiske regulatorer blevet opdaget som centrale aktører inden for mange forskellige sygdomme 1-3. Som et resultat heraf er disse enzymer primære mål for små molekyler undersøgelser og lægemiddeludvikling 4. Mange epigenetiske regulatorer har først for nylig blevet opdaget, og er stadig i færd med at blive klassificeret. Blandt disse enzymer er lysin demethylases som fjerner methylgrupper fra lysiner på histoner og andre proteiner. På grund af den hidtil ukendte arten af ​​denne klasse af enzymer, er kun få assays er blevet udviklet til at studere deres aktivitet. Det har været en vejspærring til både klassificering og high throughput undersøgelse af histon demethylases. I øjeblikket meget få demethylase analyser eksisterer. Dem, der findes tendens til at være kvalitativt i naturen og kan ikke samtidig at skelne mellem de forskellige lysin methylering (un-, mono-, di-og tri-). Massespektrometri almindeligvis anvendes til at bestemme demethylase-aktivitet, men nuværende massespektrometriske analyser gøreikke fat om differentielt methylerede peptider ionisere forskelligt. Differentiel ionisering af methylerede peptider gør sammenligning methylering vanskelig og bestemt ikke kvantitative (figur 1A). Således tilgængelige analyser er ikke optimeret til den omfattende analyse af demetylase aktivitet. Her beskriver vi en fremgangsmåde kaldet MassSQUIRM (massespektrometrisk kvantificering ved hjælp af isotop reduktiv methylering), der er baseret på reduktiv methylering af amingrupper med deutereret formaldehyd at tvinge alle lysiner er di-methyleret, hvilket gør dem alt væsentligt samme kemiske art og dermed ionisere samme (figur 1B). Den eneste kemiske forskelle efter reduktiv methylering er hydrogen, og deuterium, som ikke påvirker MALDI ionisering effektivitet. Den MassSQUIRM assay er specifik for demethylase reaktionsprodukter med un-, mono-eller di-methylerede lysiner. Analysen gælder også for lysin metyltransferaser giver samig reaktionsprodukter. Her har vi anvende en kombination af reduktiv methylering kemi og MALDI-massespektrometri til at måle aktiviteten af LSD1, en ​​lysin demethylase stand til at fjerne di-og mono-methylgrupper på et syntetisk peptidsubstrat 5. Dette assay er enkel og let modtagelige for laboratoriet med adgang til en MALDI massespektrometri i laboratoriet eller ved en proteomik-rum. Assayet har ~ 8-fold dynamikområde og er let skaleres til pladeformat 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

Vi præsenterer en fremgangsmåde til anvendelse af MALDI massespektrometri og reduktiv methylering kemi for at kvantificere ændringer i lysin methylering.

Anvendelse af MassSQUIRM for kvantitative målinger af Lysin demetylase aktivitet

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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