MassSQUIRM

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. MassSQUIRMAn assay for quantitative measurement of lysine demethylase activity Abbreviations: massSQUIRM, Mass spectrometric quantitation using isotopic reductive methylation; MAO, monoamine oxidase; PTM, post-translational modification the access of transcription machinery. 2 Some more common histone modifications include methylation, acetylation and phosphorylation. Of these common modifications, methylation is by far the most complex. Both lysine and arginine residues can be modified by mono-or di-methylation and lysine residues can be tri-methylated as well. These varying states of methylation can be associated with both active and inactive genes. The complexity and importance of methylation, on histones in particular, has stirred much interest in the enzymes capable of adding (methyltransferases) and removing (demethylases) these modifications.The first demethylase was discovered in 2004 and was termed Lysine Specific Demethylase 1 or LSD1.3 LSD1 is a flavindependent amine oxidase that can remove mono-and di-methyl marks from H3K4 primarily, H3K9 under certain conditions and some non-histone substrates such as p53. [4][5][6] It has been shown to be part of many protein complexes including CoREST, NuRD and AR/ER. 7 LSD1 is also associated with gene repression and has been suggested to be important in initiating myc-induced transcription in cancers. 3,[8][9][10][11] ©2 0 1 1 L a n d e s B i o s c i e n c e . D o n o t d i s t r i b u t e .www.landesbioscience.com Epigenetics 491

A Selective Phenelzine Analogue Inhibitor of Histone Demethylase LSD1

“…This assay is conducted for an extended time period utilizing a high LSD1 concentration, with conditions where LSD1-catalyzed demethylation of the H3-21-K4Me2 substrate nears completion, resulting in extensive conversion of the substrate to mono-and unmethylated H3-21. As reported previously, greater than 10 mM phenelzine is needed to extinguish LSD1 activity under MassSQUIRM conditions 50 . Thus, we compared 50 µM each of phenelzine and analogue 12d in an identical LSD1 inhibition MassSQUIRM assay.…”

LSD1 Histone Demethylase Assays and Inhibition

“…Several in vitro biochemical LSD1 enzymatic assays have been developed including HOOH detection through a peroxidase-coupled assay, formaldehyde detection through a formaldehyde dehydrogenase assay, radioactive measurements to monitor changes in a radiolabeled peptide substrate, and mass spectrometry analysis to measure changes in the unlabeled substrate (Blair et al, 2011; Forneris et al, 2005; Kokura, Sun, & Fang, 2015). In addition, cellular assays have been adapted to quantify LSD1-mediated effects on histone H3K4 methylation using antibody detection (Schmitt et al, 2014).…”

“…Several methods have been developed to address the methyl-Lys population states. One of these approaches involves the reductive methylation with heavy (dideuterio) formaldehyde to convert all states to the same chemical species such that there are no intrinsic ionization differences but the mass differences are detectable (Blair et al, 2011; Blair, Avaritt, & Tackett, 2012). This method, called MassSQUIRM, which uses MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) is a highly quantitative technique and has been used to assess LSD1 inhibition (Blair et al, 2011).…”

A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

“…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]. …”