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

Maile TM, Izrael-Tomasevic A, Cheung T, Guler GD, Tindell C, Masselot A, Liang J, Zhao F, Trojer P, Classon M, Arnott D - Mol. Cell Proteomics (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;

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Changes in H3K4-methylation upon siRNA-knockdown of members of the COMPASS complex in PC9 cells (left-hand panels) and HeLa cells (right hand panels).A, Western blots showing efficiency of knock-downs, H3K4me3, and loading controls. B, Relative abundance of H3K4 methyl marks for each siRNA transfection. C, Mark-by-mark changes in H3K4 methylation-levels for each siRNA transfection, expressed as log2 ratios versus siNTC control. In B and C, NTC = nontarget control, and error bars are standard deviations with n = 3.
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Figure 5: Changes in H3K4-methylation upon siRNA-knockdown of members of the COMPASS complex in PC9 cells (left-hand panels) and HeLa cells (right hand panels).A, Western blots showing efficiency of knock-downs, H3K4me3, and loading controls. B, Relative abundance of H3K4 methyl marks for each siRNA transfection. C, Mark-by-mark changes in H3K4 methylation-levels for each siRNA transfection, expressed as log2 ratios versus siNTC control. In B and C, NTC = nontarget control, and error bars are standard deviations with n = 3.

Mentions: Given the low baseline abundance of H3K4me3, demonstrating a quantitative decrease in the mark is particularly challenging. We evaluated the Prop-PIC method in this scenario in two different cell lines by depleting the protein levels of ASH2L, WDR5, or both. These are core-subunits of the COMPASS-complex, and play an essential role in the methylation of H3K4 (31). Western blots (Fig. 5A) indicated that the ASH2L and WDR5 were robustly knocked down in each cell line, although not depleted entirely. It can be seen that the H3K4me3 mark was decreased in the knock-downs, but the extent of its change is difficult to quantify. The best conditions—set 3 for the combination knock-down in PC9 cells and set 1 in HeLa cells—were chosen for mass spectrometric analysis.


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

Maile TM, Izrael-Tomasevic A, Cheung T, Guler GD, Tindell C, Masselot A, Liang J, Zhao F, Trojer P, Classon M, Arnott D - Mol. Cell Proteomics (2015)

Changes in H3K4-methylation upon siRNA-knockdown of members of the COMPASS complex in PC9 cells (left-hand panels) and HeLa cells (right hand panels).A, Western blots showing efficiency of knock-downs, H3K4me3, and loading controls. B, Relative abundance of H3K4 methyl marks for each siRNA transfection. C, Mark-by-mark changes in H3K4 methylation-levels for each siRNA transfection, expressed as log2 ratios versus siNTC control. In B and C, NTC = nontarget control, and error bars are standard deviations with n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390259&req=5

Figure 5: Changes in H3K4-methylation upon siRNA-knockdown of members of the COMPASS complex in PC9 cells (left-hand panels) and HeLa cells (right hand panels).A, Western blots showing efficiency of knock-downs, H3K4me3, and loading controls. B, Relative abundance of H3K4 methyl marks for each siRNA transfection. C, Mark-by-mark changes in H3K4 methylation-levels for each siRNA transfection, expressed as log2 ratios versus siNTC control. In B and C, NTC = nontarget control, and error bars are standard deviations with n = 3.
Mentions: Given the low baseline abundance of H3K4me3, demonstrating a quantitative decrease in the mark is particularly challenging. We evaluated the Prop-PIC method in this scenario in two different cell lines by depleting the protein levels of ASH2L, WDR5, or both. These are core-subunits of the COMPASS-complex, and play an essential role in the methylation of H3K4 (31). Western blots (Fig. 5A) indicated that the ASH2L and WDR5 were robustly knocked down in each cell line, although not depleted entirely. It can be seen that the H3K4me3 mark was decreased in the knock-downs, but the extent of its change is difficult to quantify. The best conditions—set 3 for the combination knock-down in PC9 cells and set 1 in HeLa cells—were chosen for mass spectrometric analysis.

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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;

Show MeSH