Fig. 8

Endogenous CENP-A K124 is acetylated at G1/S but monomethylated at early S and mid-S phases. a Cartoon depicting the difference between resolving modified histones on a traditional SDS-PAGE versus a long TAU (LT) or double long TAU (DLT) gel. Each additional upward shift of the histone represents an additional acetylated residue. b G1/S, early S and mid-S chromatin-bound histones were isolated, resolved on a (DLT) gel, stained with Coomassie, and endogenous CENP-A species excised for subsequent analysis by mass spectrometry. c A peptide containing acetylated lysine 124 was observed in the G1/S sample. The representative MS/MS spectrum showing CENP-A K124 acetylated in the peptide “VTLFPK(acetyl)DVQLAR” is shown on the bottom left. Location of the parent peptide ion (m/z = 714.90, charge = +2) prior to fragmentation is indicated in each spectrum with a blue diamond. Peptide fragmentation ions identified are indicated in the spectra and on the peptide sequence. The masses of ions b9, b11, y8, y9, and y10 are diagnostic of K124 acetylation. The peptide containing monomethylated lysine 124 was observed in the early S and mid-S phase—the representative MS/MS spectrum showing CENP-A K124 methylated in the peptide “VTLFPK(methyl)DVQLAR” is shown on the middle and top left. Location of the parent peptide ion (m/z = 466.60, charge = +3) prior to fragmentation is indicated in each spectrum with a blue diamond. Peptide fragmentation ions identified are indicated in the spectra and on the peptide sequence. The masses of ions b8, b10, y7, y9 and y10 are diagnostic of K124 methylation