Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications.

TitleBioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications.
Publication TypeJournal Article
Year of Publication2016
AuthorsArnaudo, AM, A Link, J, Garcia, BA
JournalACS Chem Biol
Date Published2016 Mar 18
KeywordsArginine, Chromatography, Liquid, Epitopes, Gene Expression Regulation, HeLa Cells, Histones, Humans, Isotope Labeling, Nucleosomes, Tandem Mass Spectrometry

<p>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.</p>

Alternate JournalACS Chem Biol
PubMed ID26789204
PubMed Central IDPMC4844183
Grant ListR01 AI118891 / AI / NIAID NIH HHS / United States
R01 GM110174 / GM / NIGMS NIH HHS / United States
R01AI118891 / AI / NIAID NIH HHS / United States
R01GM110174 / GM / NIGMS NIH HHS / United States