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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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Direct comparison of standard propionylation (Prop-x2) labeling and the hybrid (Prop-PIC) labeling method via LC-MS using a 1:1 mixture of labeled histone samples.A, Extracted ion chromatograms of the hydrophilic H3T3-R8 peptide (TK4QTAR) and its modified versions on C18 reverse-phase LC-MS. Retention times and relative abundances are increased by the hybrid labeling method. B, Quantitative comparison of the recoveries of each H3.1 histone tail peptide, where integrated peak areas for all modification states are combined. C, Quantitative comparison of the recoveries of the H3 T3-R8 peptide in its various modification states for the Prop-PIC versus Propx2 (integrated peak areas; n = 3, error bars are standard deviations).
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Figure 3: Direct comparison of standard propionylation (Prop-x2) labeling and the hybrid (Prop-PIC) labeling method via LC-MS using a 1:1 mixture of labeled histone samples.A, Extracted ion chromatograms of the hydrophilic H3T3-R8 peptide (TK4QTAR) and its modified versions on C18 reverse-phase LC-MS. Retention times and relative abundances are increased by the hybrid labeling method. B, Quantitative comparison of the recoveries of each H3.1 histone tail peptide, where integrated peak areas for all modification states are combined. C, Quantitative comparison of the recoveries of the H3 T3-R8 peptide in its various modification states for the Prop-PIC versus Propx2 (integrated peak areas; n = 3, error bars are standard deviations).

Mentions: Several features of the new method are immediately apparent from the extracted ion chromatograms for various H3 T3-R8 peptides (Fig. 3A). First, as expected, all detected forms of the peptide are shifted to later retention times than their Prop-x2 counterparts. Additionally, use of the Prop-PIC labeling resulted in significant increases in peak areas for all forms of the peptide, but most dramatically in the cases of H3K4me2 and -me3, which gained approximately two orders of magnitude in peak areas. The peak shape of the peptide with H3K4ac was also improved. Three technical replicates on the full procedure confirmed the consistency and superiority of the Prop-PIC protocol for H3K4 modifications (Fig. 3B). Interestingly, although not quantified in this experiment (a peak with correct mass/retention time is present but MS/MS was not recorded), H3T3 phosphorylation has been observed in the course of our COMPASS complex studies, (below; supplemental Fig. S5). This mark is associated with mitosis and has been previously identified by mass spectrometry when expedients such as phosphopeptide enrichment and/or alternative enzymatic digestions were employed.


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)

Direct comparison of standard propionylation (Prop-x2) labeling and the hybrid (Prop-PIC) labeling method via LC-MS using a 1:1 mixture of labeled histone samples.A, Extracted ion chromatograms of the hydrophilic H3T3-R8 peptide (TK4QTAR) and its modified versions on C18 reverse-phase LC-MS. Retention times and relative abundances are increased by the hybrid labeling method. B, Quantitative comparison of the recoveries of each H3.1 histone tail peptide, where integrated peak areas for all modification states are combined. C, Quantitative comparison of the recoveries of the H3 T3-R8 peptide in its various modification states for the Prop-PIC versus Propx2 (integrated peak areas; n = 3, error bars are standard deviations).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390259&req=5

Figure 3: Direct comparison of standard propionylation (Prop-x2) labeling and the hybrid (Prop-PIC) labeling method via LC-MS using a 1:1 mixture of labeled histone samples.A, Extracted ion chromatograms of the hydrophilic H3T3-R8 peptide (TK4QTAR) and its modified versions on C18 reverse-phase LC-MS. Retention times and relative abundances are increased by the hybrid labeling method. B, Quantitative comparison of the recoveries of each H3.1 histone tail peptide, where integrated peak areas for all modification states are combined. C, Quantitative comparison of the recoveries of the H3 T3-R8 peptide in its various modification states for the Prop-PIC versus Propx2 (integrated peak areas; n = 3, error bars are standard deviations).
Mentions: Several features of the new method are immediately apparent from the extracted ion chromatograms for various H3 T3-R8 peptides (Fig. 3A). First, as expected, all detected forms of the peptide are shifted to later retention times than their Prop-x2 counterparts. Additionally, use of the Prop-PIC labeling resulted in significant increases in peak areas for all forms of the peptide, but most dramatically in the cases of H3K4me2 and -me3, which gained approximately two orders of magnitude in peak areas. The peak shape of the peptide with H3K4ac was also improved. Three technical replicates on the full procedure confirmed the consistency and superiority of the Prop-PIC protocol for H3K4 modifications (Fig. 3B). Interestingly, although not quantified in this experiment (a peak with correct mass/retention time is present but MS/MS was not recorded), H3T3 phosphorylation has been observed in the course of our COMPASS complex studies, (below; supplemental Fig. S5). This mark is associated with mitosis and has been previously identified by mass spectrometry when expedients such as phosphopeptide enrichment and/or alternative enzymatic digestions were employed.

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