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

हम लिए सामूहिक spectrometric समर्थन UAMS प्रोटिओमिक्स सुविधा धन्यवाद. इस परियोजना के लिए अनुदान के एनआईएच P20RR015569 अनुदान, P20RR016460 और R01DA025755 द्वारा प्रदान किया गया.

यह प्रोटोकॉल ब्लेयर एट अल से संशोधित किया गया है 6. 1. LSD1 Demethylation परख <ol><li> 20 μL की एक अंतिम मात्रा में, 0.25 μg di मिथाइल demethylase बफर में histone H3 (ARTKme2QTARKSTGGKAPRKQLYK - बायोटिन) पेप्टाइड (50 मिमी Tris - सीएल 8.5 पीएच, 50 मिमी KCl, ...

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MassSQUIRM लाइसिन मोनो और di मेथिलिकरण में शामिल demethylases की गतिविधि के व्यापक विश्लेषण के लिए एक सस्ता और मात्रात्मक विधि है. MassSQUIRM न केवल प्रतिक्रिया के उत्पाद की लेकिन यह भी मध्यवर्ती के लिए quantitation प्रदान करता है. इ...

<table border="1"><tbody><tr><td> अभिकर्मक का नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> LSD1 </td><td> BPS बायोसाइंसेज </td><td> 50,100 </td></tr><tr><td> H3K4me2 - बायोटिन पेप्टाइड </td><td> घर में तैयार </td><td> कोई नहीं </td></tr><tr><td> Poros 20 माइक्रोन मोती R2 के </td><td> एप्लाइड Biosystems </td><td> 1-1129-06 </td></tr><tr><td> सी 18 ZipTip </td><td> Millipore </td><td> ZTC18M </td></tr><tr><td> Trifluoroacetic एसिड (TFA) </td><td> थर्मो </td><td> 28,904 </td></tr><tr><td> मिथाइल सायनाइड, एक पदार्थ जो तीन या अधिक सिगरेट रोज पीने वाले व्यक्तियों के मूत्र में अधिक मात्रा में पाया जाता है </td><td> मछुआ </td><td> A996 </td></tr><tr><td> 2,5-dihydroxybenzoic एसिड </td><td> सिग्मा </td><td> 85,707 </td></tr><tr><td> Borane dimethylamine </td><td> सिग्मा </td><td> १८०२३८ </td></tr><tr><td> isotopically भारी 2 - formaldehyde के घ </td><td> कैम्ब्रिज आइसोटोप प्रयोगशालाओं </tघ&gt; <td> DLM-805-20 </td></tr><tr><td> Tris </td><td> मछुआ </td><td> BP154 </td></tr><tr><td> KCl </td><td> मछुआ </td><td> BP366 </td></tr><tr><td> 2 MgCl </td><td> मछुआ </td><td> BP214 </td></tr><tr><td> ग्लिसरॉल </td><td> मछुआ </td><td> G33 </td></tr><tr><td> चींटी एसिड </td><td> Fluka </td><td> 06,440 </td></tr><tr><td> मेथनॉल </td><td> मछुआ </td><td> A452 </td></tr><tr><td> ना-फॉस्फेट </td><td> मछुआ </td><td> BP329 </td></tr><tr><td> SpeedVac Concentrator </td><td> विद्वान </td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF मास स्पेक्ट्रोमीटर और TOFworks सॉफ्टवेयर </td><td> PerkinElmerSciex </td><td> कोई नहीं </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

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 molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate 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

हम MALDI मास स्पेक्ट्रोमेट्री और reductive मेथिलिकरण रसायन शास्त्र का उपयोग करने के लिए lysine मेथिलिकरण में बदलाव यों के लिए एक विधि प्रस्तुत करते हैं.

MassSQUIRM की Lysine demethylase गतिविधि की मात्रात्मक मापन के लिए आवेदन

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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