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MassSQUIRM: an assay for quantitative measurement of lysine demethylase activity
Author(s) -
Avaritt Nathan L.,
Blair Lauren P.,
Tackett Alan J.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.896.2
Subject(s) - demethylase , histone , chromatin , epigenetics , lysine , methylation , biochemistry , chemistry , enzyme , in vitro , transcription (linguistics) , in vivo , dna , microbiology and biotechnology , biology , computational biology , gene , genetics , amino acid , linguistics , philosophy
In eukaryotes, DNA is wrapped around proteins called histones and condensed into chromatin. Lysine residues of the N‐terminal tails of histones can be mono‐, di‐ or tri‐methylated and these varying methylation states have been associated with different levels of gene expression. Enzymes that can add and remove these modifications are of particular interest due to their role in regulating transcription. The first demethylase discovered, LSD1 has been implicated in human cancers and contributes 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. Our results show 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. This work was supported by the following National Institutes of Health grants: NIH R01 Roadmap DA025755 (AJT), NIH INBRE P20RR016460, and NIH COBRE P20RR015569.