Name: Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity (Video) | JoVE Journal
Uploaded: 2012-03-11T14:00:00.000+00:00
Duration: 7 min 2 s
Description: Learn MassSQUIRM for quantitative lysine demethylase activity measurement using MALDI mass spectrometry and isotopic labeling. This method improves accuracy in detecting methylation changes for epigenetic research.

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5 minutes

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 μL, combine 125 ng recombinant LSD1 with 0.25 μg 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 2 hours at 37°C.</li>
	<li>To collect the peptides, add 8 μL POROS R2 20 micron beads to each sample and agitate for 15 minutes at room temperature. Prepare POROS beads by suspending 1 volume of beads with 1 volume of methanol, and then add 10 volumes of 5% formic acid / 0.2% TFA.</li>
	<li>Condition two C18 ZipTips by aspirating 20 μL of 0.1% TFA into the tip and pushing it back out with a pipettor. Do this twice with 0.1% TFA, four times with 70% acetonitrile / 0.1% TFA, and four times again with 0.1% TFA.</li>
	<li>Load the two samples (control and the demethylase reaction) onto the conditioned C18 ZipTips by pipetting the bead slurry behind the C18 resin in the ZipTips and pushing the volume through with a pipettor.</li>
	<li>Wash ZipTips twice with 20 μL of 0.1% TFA.</li>
	<li>Elute in 40 μL of 70% acetonitrile / 0.1% TFA.</li>
	<li>Lyophilize each eluant completely with a SpeedVac concentrator.</li>
</ol>
2. Reductive Methylation
This portion of the protocol is modified from Blair et al. and Rayment et al. 6,7.
<ol>
	<li>Re-suspend the demethylase reaction in 100 μL of 50 mM phosphate buffer, pH 7.4.</li>
	<li>To the demethylase reaction, add 8 μL of 15 mg/mL borane dimethylamine. Borane dimethylamine is dissolved in 50 mM phosphate buffer, pH 7.4, to give the 15 mg/mL stock solution. This should be made fresh.</li>
	<li>To the demethylase reaction, add 16 μL of 250 mM deuterated formaldehyde. The 250 mM deuterated formaldehyde is prepared by diluting the stock in H20. This should be made fresh.</li>
	<li>Incubate at 4°C for 2 hours.</li>
	<li>Repeat steps 2.2, 2.3 &amp; 2.4.</li>
	<li>Repeat step 2.2.</li>
	<li>Incubate at 4°C for ~15 hours.</li>
	<li>To the demethylase sample, add 12.5 μL of 1M Tris, pH 7.4 to quench the reductive methylation reaction.</li>
</ol>
3. MALDI Mass Spectrometry
<ol>
	<li>Add POROS R2 20 micron beads to the demethylase reaction, and collect on ZipTips as described in steps 1.3-1.6.</li>
</ol>
Note: Re-suspend the control in 100 μL of 50 mM phosphate buffer, pH 7.4 and treat as the other sample starting at step 3.1.
<ol start="2">
	<li>Elute the control and demethylase reaction samples from the ZipTips onto a MALDI sample plate using 2 μL of 33% saturated 2,5-dihydroxybenzoic acid. To prepare the 33% 2,5-dihydroxybenzoic acid, make a saturated solution of 2,5-dihydroxybenzoic acid in 70% acetonitrile / 0.1% TFA and then dilute to 33% saturated in 70% acetonitrile / 0.1% TFA. Allow the eluted samples to dry and crystallize.</li>
	<li>Collect mass spectra using a PerkinElmerSciex MALDI-prOTOF mass spectrometer or available high resolution MALDI mass spectrometer 8,9,10.</li>
	<li>View spectra and extract peak areas using PerkinElmerSciex TOFworks software or software provided by the mass spectrometer manufacturer.</li>
	<li>Verify reaction products by MS2 with an available mass spectrometric system providing tandem mass spectrometric capabilities.</li>
</ol>
4. Data Analysis
<ol>
	<li>In order to compensate for the isotopic overlap created by using heavy formaldehyde (Figure 1B), determine the peak area (A) ratio of 13C2 and 13C4 isotopes relative to the monoisotopic peak for the control sample, which has not undergone reductive methylation (Fig. 2A). 
	Eq 1: A13C2/A12C = r1 
	Eq 2: A13C4/A12C = r2 
	This will give you the ratio of peptide existing in these isotopic states (r1 &amp; r2) specific to your experiment and mass spectrometer.</li>
	<li>Use the r1 &amp; r2 values in the following formulas for quantifying the relative amount of peptide existing in each modification state in the demethylase reaction sample (Fig. 2B): 
	Eq 3: H3K4me2= A1 
	Eq 4: H3K4me = A2 - r1(A1) 
	Eq 5: H3K4 =A3 - r2(A1) - [r1(A2 - r1(A1))]</li>
</ol>
5. Representative Results
Using LSD1, we show the effectiveness of MassSQUIRM for quantifying lysine demethylation. As described in the Protocol Text section, we performed a demethylase assay with 125 ng of LSD1 and 0.25 μg di-methyl histone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotin). The control and demethylase reaction samples were subjected to reductive methylation and MALDI mass spectrometry. The control reaction that did not undergo reductive methylation showed the typical isotopic envelope for this peptide and provided peak areas for equations 1 and 2 (Figure 2A). The mass spectrum for the demethylase reaction provided peak areas for equations 3-5 (Figure 2B). Using the peak areas from the control and demethylase reaction, we determined that LSD1 demethylated the peptide to give the following reaction products: 33.7% di-methyl Lys4, 42.3% mono-methyl Lys4, and 24% un-methylated Lys 4. Refer to Blair et al. for the full analysis of LSD1 demethylase activity as well as inhibitor studies 6. Note that changing variables such as reaction time, enzyme concentration and substrate concentration will allow for an in-depth analysis of activity.
<img alt="Protein sequencing process diagram; mass spectrometry analysis; peptide fragmentation and intensity." src="/files/ftp_upload/3604/3604fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. MassSQUIRM overview. (A) An N-terminal peptide from histone H3 is shown as being un- (green), mono- (red), or di- (blue) methylated at lysine 4. Variation in the chemical composition of each peptide leads to differential ionization making quantification complex. (B) Reductive methylation converts all lysine residues to the di-methyl state which causes all peptides to ionize similarly. The use of heavy formaldehyde in the reductive methylation reaction allows retention of the identity of the original methylation. Open circles indicate light methylation while closed circles indicate heavy methylation. Modified from Blair et al. 20116.
<img alt="Mass spectrometry analysis; m/z graph showing peptide isotope labeling; chromatography result." src="/files/ftp_upload/3604/3604fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. MassSQUIRM can be used to quantify differentially methylated peptides. (A) A di-methylated synthetic histone H3 peptide (ARTKme2QTARKSTGGKAPRKQLYK-biotin) was analyzed using mass spectrometry and peak ratios relative to the monoisotopic peak were noted as r1 and r2 in equations 1 and 2. (B) The same synthetic peptide was incubated with 125 ng LSD1 in demethylase buffer for two hours at 37°C. Samples were then subjected to MassSQUIRM analysis. A mixed population of overlapping peaks represents three different methylation states as seen in Figure 1B. Areas under the monoisotopic peaks were noted as A1, A2, and A3. Peak areas were used in equations 3-5. Open circles indicate light methylation while closed circles indicate heavy methylation. Modified from Blair et al. 20116.

<ol><li>Goldberg, A. D., Allis, C. D., Bernstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics%3A+a+landscape+takes+shape%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Chromatin+modifications+and+their+function%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Epigenetic+Regulation+by+Lysine+Demethylase+5+%28KDM5%29+Enzymes+in+Cancer%5BTitle%5D)+AND+%22Cancers+%28Basel%29%22%5BJournal%5D)">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/pubmed?term=((Chemical+probes+for+histone-modifying+enzymes%5BTitle%5D)+AND+%22Nat.+Chem.+Biol%22%5BJournal%5D)">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/pubmed?term=((Histone+demethylation+mediated+by+the+nuclear+amine+oxidase+homolog+LSD1%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((MassSQUIRM%3A+An+assay+for+quantitative+measurement+of+lysine+demethylase+activity%5BTitle%5D)+AND+%22Epigenetics%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+structure+of+myosin+subfragment-1%3A+a+molecular+motor%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((CYP2E1+active+site+residues+in+substrate+recognition+sequence+5+identified+by+photoaffinity+labeling+and+homology+modeling%5BTitle%5D)+AND+%22Arch.+Biochem.+Biophys%22%5BJournal%5D)">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/pubmed?term=((Saccharomyces+cerevisiae+Yta7+Regulates+Histone+Gene+Expression%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Mol.+Cell%22%5BJournal%5D)">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>

We have nothing to disclose.

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><tbody><tr><td>LSD1</td><td>BPS Biosciences</td><td>50100</td><td></td></tr><tr><td>H3K4me2-biotin peptide</td><td>Prepared in Lab</td><td>none</td><td></td></tr><tr><td>POROS R2 20 micron beads</td><td>Applied Biosystems</td><td>1-1129-06</td><td></td></tr><tr><td>C&lt;sub&gt;18&lt;/sub&gt; ZipTip</td><td>EMD Millipore</td><td>ZTC18M</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Thermo Fisher Scientific, Inc.</td><td>28904</td><td></td></tr><tr><td>acetonitrile</td><td>Fisher Scientific</td><td>A996</td><td></td></tr><tr><td>2,5-dihydroxybenzoic acid</td><td>Sigma-Aldrich</td><td>85707</td><td></td></tr><tr><td>Borane dimethylamine</td><td>Sigma-Aldrich</td><td>180238</td><td></td></tr><tr><td>isotopically heavy &lt;em&gt;d&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt;-formaldehyde</td><td>Cambridge Isotope Laboratories</td><td>DLM-805-20</td><td></td></tr><tr><td>Tris</td><td>Fisher Scientific</td><td>BP154</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>BP366</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP214</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33</td><td></td></tr><tr><td>Formic acid</td><td>Fluka</td><td>06440</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452</td><td></td></tr><tr><td>Na-phosphate</td><td>Fisher Scientific</td><td>BP329</td><td></td></tr><tr><td>SpeedVac Concentrator</td><td>Savant</td><td>DNA110</td><td></td></tr><tr><td>MALDI-prOTOF mass spectrometer and TOFworks software</td><td>PerkinElmer, Inc.</td><td>none</td><td></td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

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.

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 ...

The mass squirm protocol aims to quantify lysine demethylase activity using isotopic methylation and maldi mass spectrometry first perform enzyme catalyzed demethylation reactions on peptides containing methylated lysine. Residues then chemically methylate the peptide products of the demethylase reaction with deuterated formaldehyde to produce uniformly methylated peptides that ionize the same way by maldi proceed to quantify each peptide species with multi to mass spectrometry and account for isotopic overlap. In order to identify relative amounts of un mono dye and trimm methylated peptides, the mass squirm results determine relative amounts of each demethylase reaction product based on changes in mass.The primary advantage of this technique over existing methods like routine moldy mass spectrometry is that mass score accounts for differences in ionization between differently methylated peptides. We first had the idea for this approach when we developed a method to measure histone acetylation levels using isotopically heavy acetic anhydride. The challenge to this technique is extracting peak area data, which can be complicated by overlap of peptide isotopic envelopes.For the demethylase reaction combined 125 nanograms of recombinant lysine specific demethylase one enzyme with 0.25 micrograms of biotinylated dimethyl hisone H three peptide in a final volume of 20 microliters demethylase buffer. Also prepare a control sample containing 0.25 micrograms peptide without the LSD one enzyme incubate the reaction and control tubes for two hours at 37 degrees Celsius. Next to prepare R two 20 micron porous beads mix equal volumes of beads and methanol.Then add 10 volumes of TFA formic acid, add eight microliters of porous beads to each sample and agitate for 15 minutes at room temperature. Meanwhile, condition two C 18 zip tips. Pipetting twice with 20 microliters of 0.1%TFA four times with 70%aceto nitrile 0.1%TFA, and four times again with 0.1%TFA.Next, load the two samples onto the conditioned zip tips. With a pipetter, push the volume through the C 18 resin. Repeat until total volume of both samples has been pushed through the zip tips.Then wash the zip tips twice with 20 microliters of 0.1%TFA elute samples in 40 microliters illusion buffer proceed to dry eent completely with a speed vac concentrator. First resuspend the sample in 100 microliters of 50 millimolar phosphate buffer pH 7.4. Then add eight microliters of bore dimethyl amine and 16 microliters of derated formaldehyde.Incubate at four degrees Celsius for two hours. Repeat the reductive methylation by adding boring dimethyl lamine and deuterated formaldehyde and incubate for two hours. The third time, add boring dimethyl lamine and incubate at four degrees Celsius for 15 hours.Quench the reaction with 12.5 microliters of one molar tris. pH 7.4 proceed to dry eent completely with a speed vac concentrator. First Resus, suspend the control and demethylase reaction samples in 100 microliters of 50 millimolar phosphate buffer pH 7.4.Now add eight microliters porous beads to the control and demethylase reaction samples and collect on zip tips as shown earlier. Elude in two microliter volumes onto a MALDI sample plate and allow to dry at room temperature to crystallization. Proceed to analyze samples by mass spectrometry.Open the spectro files in to work software and export to M over Z software. Further verify reaction products by tandem mass spectrometry. First, determine the peak area ratio of carbon 13 C two and carbon 13 C four isotopes relative to the mono isotopic peak for the control sample.Use these calculated ratios to quantify the relative amount of peptide existing in each modification. State of the demethylase reaction sample. The LSD one enzyme can be effectively used in mass squirm quantification of lysine demethylation.Since the control reaction does not undergo reductive methylation, it indicates the typical isotopic envelope for this peptide Resulting peak areas determine background ratios of peptide existing in carbon 13 C two and carbon 13 C four. Isotopic states, peak areas from the mass spectrum for the control reaction allow quantification of the relative amount of peptide existing in each modification state. In the demethylase reaction, the peak areas from these control and demethylase reactions determine 33.7%dimethyl lysine 4 42 0.3%monomethyl lysine four and 24%unmethylated lysine four.This mass squam approach offers key insights into the field of histone epigenetics, such as how demethylation of histone lysine residues contributes to various diseases. While this methylation assay has been applied to study demethylases, it can also be used to study methyl transferases as well.

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Learn MassSQUIRM for quantitative lysine demethylase activity measurement using MALDI mass spectrometry and isotopic labeling. This method improves accuracy in detecting methylation changes for epigenetic research.

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

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