Extended Data Fig. 7: Characterization of H3 enzymatic methylation by Dim5.

Methylation of H3 was followed by EST-based LC–MS/MS analysis of the intact proteins. a, Charge state envelope of untreated (left) and methylated (right) H3. The bottom panel, focused on a single charge state, shows disappearance of the starting material and the formation of higher mass species, spaced 14 Da apart. b, ETD-based mass spectrometry of precursor ions corresponding to unmethylated H3 (top), H3K9me3 (middle) and H3K9me3-K18me3 (bottom). The rationale for these assignments is based on the precursor mass values and by the product ions. The z-ions (purple) do not change between the three spectra, whereas the pattern of c-ions (blue) are mass shifted by 42 Da and charge-state shifted by 1+ at C9 between the top and middle or bottom panels, and again at C18 between the middle and bottom panels. These assignments are further validated by bottom-up proteomics analysis of Lys-C-digested samples (not shown). c, The precursor ion spectra in a were deconvoluted using Xtract, which models both the charge states and the isotope distributions. Deconvoluted MH⁺ values are consistent with multiple methylation states (owing to the difficulty of modelling isotope distributions from large proteins, particularly as there is some underlying oxidation, Xtract sometimes picks the wrong monoisotope). Deconvoluted intensities show that the enzymatically treated sample (right) contains no notable unmethylated H3 or mono- and di-methylated H3K9. Although 100% of analysed sample is tri-methylated at H3K9, additional methylations occur at H3K18 as noted above. The sample analysed here was used to carry out all the experiments to minimize variability.