Quantitative proteomics has focused heavily on correlating protein abundances, 
ratios, and dynamics by developing methods that are protein expression-centric 
(e.g. isotope coded affinity tag, isobaric tag for relative and absolute 
quantification, etc.). These methods effectively detect changes in protein 
abundance but fail to provide a comprehensive perspective of the diversity of 
proteins such as histones, which are regulated by post-translational 
modifications. Here, we report the characterization of modified forms of HeLa 
cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass 
spectrometric approach coupled with two dimensions of liquid chromatography. 
This enhanced dynamic range enabled the precise characterization and 
quantitation of 42 forms uniquely modified by combinations of methylation and 
acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, 
and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative 
analyses revealed distinct trends in acetylation site occupancy depending on 
Lys-20 methylation state. Because both modifications are dynamically regulated 
through the cell cycle, we simultaneously investigated acetylation and 
methylation kinetics through three cell cycle phases and used these data to 
statistically assess the robustness of our quantitative analysis. This work 
represents the most comprehensive analysis of histone H4 forms present in human 
cells reported to date.