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1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences
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We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.
Blair, L. P., Avaritt, N. L., Tackett, A. J. Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity. J. Vis. Exp. (61), e3604, doi:10.3791/3604 (2012).
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.
This protocol is modified from Blair et al. 6.
1. LSD1 Demethylation Assay
2. Reductive Methylation
This portion of the protocol is modified from Blair et al. and Rayment et al. 6,7.
3. MALDI Mass Spectrometry
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.
4. Data Analysis
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.
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.
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.
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.
We have nothing to disclose.
We thank the UAMS Proteomics Facility for mass spectrometric support. Funding for this project was provided by NIH grants P20RR015569, P20RR016460 and R01DA025755.
|H3K4me2-biotin peptide||Prepared in Lab||none|
|POROS R2 20 micron beads||Applied Biosystems||1-1129-06|
|C18 ZipTip||EMD Millipore||ZTC18M|
|Trifluoroacetic acid (TFA)||Thermo Fisher Scientific, Inc.||28904|
|isotopically heavy d2-formaldehyde||Cambridge Isotope Laboratories||DLM-805-20|
|MALDI-prOTOF mass spectrometer and TOFworks software||PerkinElmer, Inc.||none|
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