Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.
Bottom Line: 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.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.
Affiliation: From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;Show MeSH
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Mentions: Cells derived from non small cell lung cancer (PC9) were treated in order to assess the activity and specificity of several putative KDM-inhibitors, and subjected to mass spectrometric analysis. Fig. 4 illustrates the results for an inactive control compound (compound A) and four active compounds (B, C, D, and E) to assess their potency and selectivity. A heat-map representation shows how each of 25 marks changes upon drug treatment relative to cells treated with DMSO diluent alone (Fig. 4A). All of the active compounds increased H3 K4-trimethylation to some degree, and in a dose dependent manner for compounds C and D, which were each tested at two concentrations (Fig. 4B). Each sample also contained stable isotope-labeled histones from untreated PC9 cells as internal standards. These constitute technical replicates and should in principle have identical abundances in all samples. Their variability therefore sets a lower bar for the reliability of quantifying each observed histone mark. In this case the 95% confidence interval (n = 10) for H3K4me3 is ± 0.2 log units; smaller changes are unlikely to be significant. Western blots of the same samples for H3K4me3 (Fig. 4C) are consistent with the mass spectrometric results, but are much less quantitative. Compound B showed the least change in H3K4-trimethylation, and was also the least selective in terms of changing modification levels at individual histone lysine residues. Indeed, the most dramatic effects were decreases in monomethylation at H3K18 as well as dimethylation at both H3K4 and H3K36, making this compound unattractive for further investigation. The other three compounds had much cleaner profiles, with small, across-the board increases in H3K4-dimethylation, and more variable, but relatively small H3K9 di- and trimethylation, and H3K36 trimethylation. Interestingly, increases of acetylation at H3K9 were observed for compounds C, D, and E, and were dose-dependent for compounds C and D. More subtle increases in acetylation at H3K14 and H3K18 were found for compounds C and E. These might be off-target effects of the drugs, or secondary effects of chromatin remodeling that follow from changes in H3K4-methylation.
Affiliation: From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;