cb5b00816_si_003.pdf (257.31 kB)
Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications
journal contributionposted on 2016-02-10, 14:52 authored by Anna M. Arnaudo, A. James Link, Benjamin A. Garcia
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 13C6,15N4 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.