title: Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications.
abstract: The nucleosome is an octamer containing DNA wrapped around one histone H3-H4 tetramer and two histone H2A-H2B dimers. Within the nucleosome, histones are decorated with post-translational modifications. Previous studies indicate that the H3-H4 tetramer is conserved during DNA replication, suggesting that old tetramers serve as a template for the modification of newly synthesized tetramers. Here, we present a method that merges bioorthogonal chemistry with mass spectrometry for the study of modifications on newly synthesized histones in mammalian cells. HeLa S3 cells are dually labeled with the methionine analog azidohomoalanine and heavy (13)C6,(15)N4 isotope labeled arginine. Heavy amino acid labeling marks newly synthesized histones while azidohomoalanine incorporation allows for their isolation using bioorthogonal ligation. Labeled mononucleosomes were covalently linked via a copper catalyzed reaction to a FLAG-GGR-alkyne peptide, immunoprecipitated, and subjected to mass spectrometry for quantitative modification analysis. Mononucleosomes containing new histones were successfully isolated using this approach. Additionally, the development of this method highlights the potential deleterious effects of azidohomoalanine labeling on protein PTMs and cell cycle progression, which should be considered for future studies utilizing bioorthogonal labeling strategies in mammalian cells.
authors.affiliation: Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, United States.|Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Arnaudo
authors.affiliation: Departments of Chemical and Biological Engineering, Princeton University , Princeton, New Jersey 08544, United States.
authors.forename: A James
authors.initials: AJ
authors.lastname: Link
authors.affiliation: Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
mesh_terms: D001120:Arginine; D002853:Chromatography, Liquid; D000939:Epitopes; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006657:Histones / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D009707:Nucleosomes; D053719:Tandem Mass Spectrometry
chemical_list: D000939:Epitopes; D006657:Histones; D009707:Nucleosomes; D001120:Arginine