Supplementary Figure 6: Ezh2 mediates trimethylation of talin1 at Lys2454.
ETD-MS/MS spectra were recorded by Orbitrap Elite in the supplemental activation ETD mode. MS/MS spectra of the unmodified peptide (Red) and the K15Me3 modified peptides (Blue) were displayed in the stack plot with Y-offset of 10. The supplemental activation ETD fragmentation of peptide produced mainly c and z· ions, with some b and y ions. The ions of the small fragments of the two peptides that do not contain the K15Me3 (talin1-K2454) have the same masses (m/z) and were labeled in black. The major c and z· ions of larger fragments that contain the K15 (Red) from the unmodified peptide or K15Me3 (Blue) from the modified peptide were labeled. The spectra showed a systematic 42 Da shift in the fragment containing the K15Me3 modification. The K15 modification site is sandwiched by the series of c and z· ions, indicating unambiguous assignment of the K15Me3 modification. Both peptides have N22 deamidation that are commonly generated during peptide synthesis or sample handling steps. The detailed fragment ions assignment of the supplemental activation ETD-MS/MS spectrum of the unmodified and methylated peptide by Mascot search engine version 2.4 was shown in Supplementary Tables 1 and 2. As a mixture of unmodified, oxidized, deamidated, and/or K15-trimethylated peptides were generated in the in-vitro methylation process, it is impractical to use extracted ion chromatogram method to integrate peak areas of various modified peptides for label-free quantitation. Spectral counting method was used to determine the K15-trimethylation efficiency by the Ezh2. We counted all MS/MS spectra matched to the peptides with no modification, methionine oxidation, deamidation and/or trimethylation. In total, there are 81 MS/MS spectra matched to the peptides. Among them, 31 MS/MS spectra were identified with trimethylation. In summary, the methylation efficiency of the Ezh2 on this peptide is determined to be 37% (details see corresponding Source Data file).
