Article

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

ABSTRACT

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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    • "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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