MassSQUIRM की Lysine demethylase गतिविधि की मात्रात्मक मापन के लिए आवेदन
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the reagent | Company | Catalogue number |
| LSD1 | BPS Biosciences | 50100 |
| H3K4me2-biotin peptide | prepared in-house | none |
| POROS R2 20 micron beads | Applied Biosystems | 1-1129-06 |
| C18 ZipTip | Millipore | ZTC18M |
| Trifluoroacetic acid (TFA) | Thermo | 28904 |
| acetonitrile | Fisher | A996 |
| 2,5-dihydroxybenzoic acid | Sigma | 85707 |
| Borane dimethylamine | Sigma | 180238 |
| isotopically heavy d2-formaldehyde | Cambridge Isotope Laboratories | DLM-805-20 |
| Tris | Fisher | BP154 |
| KCl | Fisher | BP366 |
| MgCl2 | Fisher | BP214 |
| Glycerol | Fisher | G33 |
| Formic acid | Fluka | 06440 |
| Methanol | Fisher | A452 |
| Na-phosphate | Fisher | BP329 |
| SpeedVac Concentrator | Savant | DNA110 |
| MALDI-prOTOF mass spectrometer and TOFworks software | PerkinElmerSciex | none |
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