Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Summary

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

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.

Protocol

This protocol is modified from Blair et al. ⁶.

1. LSD1 Demethylation Assay

1. 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.
2. Incubate for 2 hours at 37°C.
3. To collect the peptides, add 8 μL POROS R2 20 micron beads to each sample and agitate for 15 minutes at room temperature. Prepare....

Discussion

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.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LSD1	BPS Biosciences	50100
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
acetonitrile	Fisher Scientific	A996
2,5-dihydroxybenzoic acid	Sigma-Aldrich	85707
Borane dimethylamine	Sigma-Aldrich	180238
isotopically heavy d2-formaldehyde	Cambridge Isotope Laboratories	DLM-805-20
Tris	Fisher Scientific	BP154
KCl	Fisher Scientific	BP366
MgCl2	Fisher Scientific	BP214
Glycerol	Fisher Scientific	G33
Formic acid	Fluka	06440
Methanol	Fisher Scientific	A452
Na-phosphate	Fisher Scientific	BP329
SpeedVac Concentrator	Savant	DNA110
MALDI-prOTOF mass spectrometer and TOFworks software	PerkinElmer, Inc.	none