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Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications
journal contribution
posted on 2016-02-10, 14:52 authored by Anna M. Arnaudo, A. James Link, Benjamin A. GarciaThe
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.