Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.

Autor:	Maile TM; From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;, Izrael-Tomasevic A; From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;, Cheung T; From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;, Guler GD; §Cancer Targets Department, Genentech, Inc., South San Francisco, California 94080;, Tindell C; §Cancer Targets Department, Genentech, Inc., South San Francisco, California 94080;, Masselot A; ¶Department of Bioinformatics and Computational Biology, Genentech, Inc., South San Francisco, California 94080;, Liang J; ‖Discovery Chemistry Department, Genentech, Inc., South San Francisco, California 94080;, Zhao F; *Biology Department, Constellation Pharmaceuticals, Inc., Cambridge, Massachusetts 02142., Trojer P; *Biology Department, Constellation Pharmaceuticals, Inc., Cambridge, Massachusetts 02142., Classon M; §Cancer Targets Department, Genentech, Inc., South San Francisco, California 94080;, Arnott D; From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080; arnott@gene.com.
Jazyk:	angličtina
Zdroj:	Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2015 Apr; Vol. 14 (4), pp. 1148-58. Date of Electronic Publication: 2015 Feb 13.
DOI:	10.1074/mcp.O114.046573
Abstrakt:	Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a "one-pot" hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5. (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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