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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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Sources of discrepancy in H3K4 quantitation.A, Equimolar mixture of isotope-labeled, propionylated H3 T3-R8 peptide in eight modification states, injected in the milieu of endogenous histones from 293T cells. B, Time course of digestion releasing propionylated H3 T3-R8 from propionylated H3 A1-R17, where K4 is trimethylated. C, Relative ionization efficiencies of the propionylated H3 T3-R8 peptide in its various modified forms. D, Recovery of H3 T3-R8 peptides following StageTip C18 cleanup. In panels A, C, D, all abundances are expressed relative to the unmodified peptide.
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Figure 1: Sources of discrepancy in H3K4 quantitation.A, Equimolar mixture of isotope-labeled, propionylated H3 T3-R8 peptide in eight modification states, injected in the milieu of endogenous histones from 293T cells. B, Time course of digestion releasing propionylated H3 T3-R8 from propionylated H3 A1-R17, where K4 is trimethylated. C, Relative ionization efficiencies of the propionylated H3 T3-R8 peptide in its various modified forms. D, Recovery of H3 T3-R8 peptides following StageTip C18 cleanup. In panels A, C, D, all abundances are expressed relative to the unmodified peptide.

Mentions: Eight synthetic peptides corresponding to histone H3 residues 1–17 (ARTKQTARKSTGGKAPR) were synthesized, identical except for their modification states at lysine-4 (unmodified, acetylated, mono-/di- and tri-methylated), and/or phosphorylated at threonine-6. Each of these peptides was treated according to published protocols (10) with isotopically labeled (d10) propionic anhydride to block the peptide N terminus and the unmodified lysines. Monomethyl K4 was also thereby converted to monomethyl, propionyl K4. Trypsin digestion was followed by a second round of propionylation to block the new peptide N termini. These peptides, in nominally equimolar ratios, were mixed with endogenous histones from 293T cells that had been prepared identically except without stable isotope labeling, and analyzed by capillary LC-MS/MS on an Orbitrap mass spectrometer. When the peak areas of the synthetic peptides - distinguishable by their stable isotope labels - were extracted, it was evident that the “recoveries” of differently modified forms were quite different (Fig. 1A). Whereas most versions of the peptide were detected with abundance equal to or greater than the unmodified peptide, the dimethyl, trimethyl, phosphoryl, and dimethyl/phosphoryl forms were all below 10% abundance relative to their unmodified counterpart. Although different peptides are not expected to have identical responses in the mass spectrometer, such large and selective discrimination against specific modified forms could confound quantitative experiments, particularly where the modified form is of low natural abundance to begin with, as is the cases of H3K4me2 and -me3.


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)

Sources of discrepancy in H3K4 quantitation.A, Equimolar mixture of isotope-labeled, propionylated H3 T3-R8 peptide in eight modification states, injected in the milieu of endogenous histones from 293T cells. B, Time course of digestion releasing propionylated H3 T3-R8 from propionylated H3 A1-R17, where K4 is trimethylated. C, Relative ionization efficiencies of the propionylated H3 T3-R8 peptide in its various modified forms. D, Recovery of H3 T3-R8 peptides following StageTip C18 cleanup. In panels A, C, D, all abundances are expressed relative to the unmodified peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Sources of discrepancy in H3K4 quantitation.A, Equimolar mixture of isotope-labeled, propionylated H3 T3-R8 peptide in eight modification states, injected in the milieu of endogenous histones from 293T cells. B, Time course of digestion releasing propionylated H3 T3-R8 from propionylated H3 A1-R17, where K4 is trimethylated. C, Relative ionization efficiencies of the propionylated H3 T3-R8 peptide in its various modified forms. D, Recovery of H3 T3-R8 peptides following StageTip C18 cleanup. In panels A, C, D, all abundances are expressed relative to the unmodified peptide.
Mentions: Eight synthetic peptides corresponding to histone H3 residues 1–17 (ARTKQTARKSTGGKAPR) were synthesized, identical except for their modification states at lysine-4 (unmodified, acetylated, mono-/di- and tri-methylated), and/or phosphorylated at threonine-6. Each of these peptides was treated according to published protocols (10) with isotopically labeled (d10) propionic anhydride to block the peptide N terminus and the unmodified lysines. Monomethyl K4 was also thereby converted to monomethyl, propionyl K4. Trypsin digestion was followed by a second round of propionylation to block the new peptide N termini. These peptides, in nominally equimolar ratios, were mixed with endogenous histones from 293T cells that had been prepared identically except without stable isotope labeling, and analyzed by capillary LC-MS/MS on an Orbitrap mass spectrometer. When the peak areas of the synthetic peptides - distinguishable by their stable isotope labels - were extracted, it was evident that the “recoveries” of differently modified forms were quite different (Fig. 1A). Whereas most versions of the peptide were detected with abundance equal to or greater than the unmodified peptide, the dimethyl, trimethyl, phosphoryl, and dimethyl/phosphoryl forms were all below 10% abundance relative to their unmodified counterpart. Although different peptides are not expected to have identical responses in the mass spectrometer, such large and selective discrimination against specific modified forms could confound quantitative experiments, particularly where the modified form is of low natural abundance to begin with, as is the cases of H3K4me2 and -me3.

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