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.
Highlights
These proteins are heavily modified, with combinations of these enzymatic modifications thought to form a “histone code” orchestrating epigenetic processes such as long-term gene silencing and gene activation [1], higher level chromatin packaging [2], and DNA repair mechanisms [3]. All of these activities change with relation to the cell cycle, a sequence of events during which a cell commits to DNA replication (G1), replicates its DNA (S), prepares for mitosis (G2), and undergoes cell division (M) [4]
It has been asserted that methylation of lysine 9 in human H3 is a mark for heterochromatin by interacting with heterochromatin-associated protein 1 [11, 12]
Our laboratories have successfully utilized a Hydrophilic interaction chromatography (HILIC)/Top Down mass spectrometry (TDMS) approach to identify more than 150 different combinations of post-translational modifications on histone H3.2 [26]
Summary
Histone Preparation: Reverse Phase and Hydrophilic Interaction Liquid Chromatography—RP-HPLC-purified H4 was prepared by chromatography of ϳ100 g of crude HeLa S3 histone protein (with or without prior oxidation as described previously [28, 29]). Quantitation of Modified Forms of Intact H4—The molecules remain intact so that all possible combinations of modifications are present when analyzed by TDMS With this in mind, the HILIC chromatogram was integrated, and each fraction was assigned a percent area relative to the entire elution area of H4. ECD generated 1ϩ, 2ϩ, and 3ϩ fragment ions, from which the 13C0, 13C1, and 13C2 (when observed) isotopes were integrated and used to calculate the FIRRs. Software—A Web-based software and data base suite, ProSight PTM [21, 30], was used to accelerate the characterization of histone H4 protein forms as previously reported [21]. The chromatographic area of this peak, representing an estimated ϳ0.7% of the most abundant peak, was subtracted of the whole and removed from further calculations and analysis
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