Middle-Down Characterization of the Cell Cycle Dependence of Histone H4 PTMs and Proteoforms

In the present study, a middle-down approach is employed for the identification, characterization, and quantitation of the PTMs and proteoforms of histone H4 in two breast cancer cell lines. Our experimental strategy not only preserves the combinatorial attributes of existing PTMs, but also enhances the separation ability and reduces the complexity of downstream data analysis. The fluctuations in the relative abundances of common modifications throughout the cell cycle, including phosphorylation, acetylation, and methylation, suggest that they play significant roles at different stages of the cell cycle in breast cancer and could participate in carcinogenesis. In summary, our analysis advances the understanding and elucidation of cancer epigenetics at the single-molecule level, and lays the foundation for further study of histones or other proteins.