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: A new protocol was therefore implemented (Fig. 2A and supplemental Fig. S2), similar to the “standard” protocol in that predigestion propionylation was used to block unmodified and mono-methyl lysine, but differing in that postdigestion labeling is done with PIC, resulting in a single PIC label at the N terminus of each tryptic peptide. Another difference is that propionylation is now performed in aqueous solution at pH 8.5, buffered by triethylammonium bicarbonate, with excess propionic anhydride quenched stoichiometrically in situ, allowing the procedure to be performed in a single tube without vacuum drying to remove reagents between steps. Neither is it necessary to repeatedly check and adjust pH, as is necessary during the multiple propionylation reactions of the standard protocol, resulting in a significant reduction in the number of sample handling steps at which losses might be accrued. The entire protocol can be performed in less than a day (four hours of which are incubation with trypsin), so that samples can be prepared and loaded onto an autosampler for overnight LC-MS/MS. Ionization efficiencies and reverse-phase trapping column recoveries were determined for the propionylated/PIC labeled peptides as before (Figs. 2B and 2C). Relative ionization efficiencies among the various H3 K4 modifications were not significantly changed, but the recoveries of K4me3 and K4me2/T6 phosphoryl peptides were dramatically improved. Indeed, if the peptide abundances were corrected for ionization efficiency, the recoveries of the H3 T3-R8 peptide in all modified forms would be approximately equal. N-terminal PIC labeling does not strongly direct peptide fragmentation patterns under collision-induced dissociation, but a subtle decrease in b-ion abundances can be discerned, particularly in the first ions of the series. Interestingly, b1 ions are not normally observed in collision-induced dissociation of peptides with free N termini, but are prominent in the product ion spectra of peptides N-terminally modified by either propionylation or PIC, reflecting the presumed mechanism of low energy fragmentation (25). Indeed, the combination of Lys-propionylation and N-terminal PIC labeling produces other low mass ions in HCD that are diagnostic of several modifications including propionyl-Lys (m/z 140.107), [monomethyl, propionyl]-Lys (m/z 171.14), and N-terminal lysine with propionyl, [monomethyl, propionyl], dimethyl, and acetyl modifications at the epsilon amine (m/z 276.17, 318.18, 290.15, respectively). The slight decrease in b-ion abundances with PIC labeling seems correlated with higher abundances of these diagnostic ions, although the matter has not been explored systematically.
Affiliation: From the ‡Protein Chemistry Department, Genentech Inc., South San Francisco, California 94080;