title: Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.
abstract: 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.
authors.affiliation: Center for Biophysics and Computational Biology, Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Pesavento
authors.forename: Courtney R
authors.initials: CR
authors.lastname: Bullock
authors.forename: Richard D
authors.initials: RD
authors.lastname: LeDuc
authors.forename: Craig A
authors.initials: CA
authors.lastname: Mizzen
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D002453:Cell Cycle / Q000502:physiology*; D002853:Chromatography, Liquid / Q000379:methods; D006367:HeLa Cells; D006657:Histones / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D040901:Proteomics / Q000379:methods
chemical_list: D006657:Histones