title: Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers.
abstract: Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein-protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides.
authors.affiliation: Department of Systems Biology and Medicine, Research Center for Advanced Science and Technology,  The University of Tokyo; The Translational Systems Biology and Medicine Initiative Center for Disease Biology and Integrative Medicine,  Faculty of Medicine, University of Tokyo.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Systems Biology and Medicine, Research Center for Advanced Science and Technology,  The University of Tokyo; The Translational Systems Biology and Medicine Initiative Center for Disease Biology and Integrative Medicine,  Faculty of Medicine, University of Tokyo.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Chikaoka
authors.affiliation: Department of Chemistry and Biotechnology, The University of Tokyo.
authors.forename: Gosuke
authors.initials: G
authors.lastname: Hayashi
authors.affiliation: Department of Chemistry and Biotechnology, The University of Tokyo.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Sakamoto
authors.affiliation: Department of Systems Biology and Medicine, Research Center for Advanced Science and Technology,  The University of Tokyo.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Yamamoto
authors.affiliation: Radioisotope Center, The University of Tokyo.
authors.forename: Akira
authors.initials: A
authors.lastname: Sugiyama
authors.affiliation: Department of Systems Biology and Medicine, Research Center for Advanced Science and Technology,  The University of Tokyo.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo.
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Okamoto
authors.affiliation: Department of Systems Biology and Medicine, Research Center for Advanced Science and Technology,  The University of Tokyo; The Translational Systems Biology and Medicine Initiative Center for Disease Biology and Integrative Medicine,  Faculty of Medicine, University of Tokyo.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawamura
keywords: ETD; cell cycle; cross-linking; epigenetics; histone
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