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Post-translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle-down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) for on-line liquid chromatography-mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to unambiguously distinguish between acetylation and tri-methylation (∆m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono- and di-methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle-dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Histone posttranslational modifications (PTMs) help regulate DNA templated processes; however, relatively little work has unbiasedly explored the single-molecule combinations of histone PTMs, their dynamics on short timescales, or how these preexisting histone PTMs modulate further histone modifying enzyme activity. We use quantitative top down proteomics to unbiasedly measure histone H4 proteoforms (single-molecule combinations of PTMs) upon butyrate treatment. Our results show that histone proteoforms change in cells within 10 minutes of application of sodium butyrate. Cells recover from treatment within 30 minutes after removal of butyrate. Surprisingly, K20me2 containing proteoforms are the near-exclusive substrate of histone acetyltransferases upon butyrate treatment. Single-molecule hierarchies of progressive PTMs mostly dictate the addition and removal of histone PTMs (K16ac > K12ac ≥ K8ac > K5ac, and the reverse on recovery). This reveals the underlying single-molecule mechanism that explains the previously reported but indistinct and unexplained patterns of H4 acetylation. Thus, preexisting histone PTMs strongly modulate histone modifying enzyme activity and this suggests that proteoform constrained reaction pathways are crucial mechanisms that enable the long-term stability of the cellular epigenetic state.