We thank the UAMS Proteomics Facility for mass spectrometric support. Funding for this project was provided by NIH grants P20RR015569, P20RR016460 and R01DA025755.

This protocol is modified from Blair et al. 6.
1. LSD1 Demethylation Assay
<ol>
	<li>In a final volume of 20 &mu;L, combine 125 ng recombinant LSD1 with 0.25 &mu;g di-methyl histone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotin) in demethylase buffer (50 mM Tris-Cl pH 8.5, 50 mM KCl, 5 mM MgCl2, 5% glycerol). Additionally, perform a peptide alone control with no enzyme. The control is for section 3 and does not need reductive methylation.</li>
	<li>Incubate for ...

<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 is an inexpensive and quantitative method for comprehensive analysis of the activity of lysine demethylases involved in mono- and di-methylation. MassSQUIRM offers quantitation not only of the product of the reaction but also for the intermediates. This assay can be used as a powerful tool in studying the mechanism of LSD1 and other histone demethylases. It will also be useful for classifying many newly discovered lysine demethylase enzymes such as PHF8 and could be used for certain methyltransferase enzymes.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>LSD1</td>
			<td>BPS Biosciences</td>
			<td>50100</td>
		</tr>
		<tr>
			<td>H3K4me2-biotin peptide</td>
			<td>prepared in-house</td>
			<td>none</td>
		</tr>
		<tr>
			<td>POROS R2 20 micron beads</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>Trifluoroacetic acid (TFA)</td>
			<td>Thermo</td>
			<td>28904</td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Fisher</td>
			<td>A996</td>
		</tr>
		<tr>
			<td>2,5-dihydroxybenzoic acid</td>
			<td>Sigma</td>
			<td>85707</td>
		</tr>
		<tr>
			<td>Borane dimethylamine</td>
			<td>Sigma</td>
			<td>180238</td>
		</tr>
		<tr>
			<td>isotopically heavy d2-formaldehyde</td>
			<td>Cambridge Isotope Laboratories</td>
			<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>Formic acid</td>
			<td>Fluka</td>
			<td>06440</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A452</td>
		</tr>
		<tr>
			<td>Na-phosphate</td>
			<td>Fisher</td>
			<td>BP329</td>
		</tr>
		<tr>
			<td>SpeedVac Concentrator</td>
			<td>Savant</td>
			<td>DNA110</td>
		</tr>
		<tr>
			<td>MALDI-prOTOF mass spectrometer and TOFworks software</td>
			<td>PerkinElmerSciex</td>
			<td>none</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

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

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 vistas.  University of Arkansas for Medical Sciences. Se presenta un método para el uso de espectrometría de masas MALDI y la química de la metilación reductiva para cuantificar los cambios en la metilación de la lisina.