MassSQUIRM An assay for quantitative measurement of lysine demethylase activity

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Epigenetics: official journal of the DNA Methylation Society (Impact Factor: 4.78). 04/2011; 6(4):490-9. DOI: 10.4161/epi.6.4.14531
Source: PubMed


In eukaryotes, DNA is wrapped around proteins called histones and is condensed into chromatin. Post-translational modification of histones can result in changes in gene expression. One of the most well-studied histone modifications is the methylation of lysine 4 on histone H3 (H3K4). This residue can be mono-, di- or tri-methylated and these varying methylation states have been associated with different levels of gene expression. Understanding exactly what the purpose of these methylation states is, in terms of gene expression, has been a topic of much research in recent years. Enzymes that can add (methyltransferases) and remove (demethylases) these modifications are of particular interest. The first demethylase discovered, LSD1, is the most well-classified and has been implicated in contributing to human cancers and to DNA damage response pathways. Currently, there are limited methods for accurately studying the activity of demethylases in vitro or in vivo. In this work, we present MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation), a quantitative method for studying the activity of demethylases capable of removing mono- and di-methyl marks from lysine residues. We focus specifically on LSD1 due to its potential as a prime therapeutic target for human disease. This quantitative approach will enable better characterization of the activity of LSD1 and other chromatin modifying enzymes in vitro, in vivo or in response to inhibitors.

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Available from: Rong Huang, Oct 04, 2015
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    • "There have been studies that attempted to quantify methylation by direct spotting, but to our knowledge no studies have reported kinetic analyses for peptide substrates, most likely because of the limited sensitivity. The MassSQUIRM technique has been developed to utilize isotopic enrichment to increase sensitivity, but requires deuterated formaldehyde to generate the substituted peptides [31]. "
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    ABSTRACT: Protein methylation and acetylation play an important role in biological processes, and misregulation of these modifications are involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic protein methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 femtomoles. This is achieved by increasing the signal-to-noise ratio through addition of NH4H2PO4 to the matrix solution and reduction of the matrix CHCA concentration to 2 mg/mL. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted for the investigation of the activity of any protein methyltransferases and acetyltransferases. Copyright © 2015 Elsevier Inc. All rights reserved.
    Analytical Biochemistry 03/2015; 478:59-64. DOI:10.1016/j.ab.2015.03.007 · 2.22 Impact Factor
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    • "Some of the limitations in drug discovery thus far have been the lack of appropriate readouts for demethylase activity and the similarity of mechanism between all proteins of this class. Recently, more accurate demethylase assays have been developed and high throughput screening techniques have been optimized for identifying specific inhibitors of these enzymes [128,129]. These inhibitors/epigenetic drugs will have the potential to reprogram cancer cells or cancer stem cells with the same gusto as the HDAC inhibitors and could have a revolutionary effect on the future of cancer treatment. "
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    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).
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