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|Title:||Development of a Bioorthogonal Chemistry Based Method for the Isolation of New Histones and the Study of PTM Propagation|
|Authors:||Arnaudo, Anna Marie|
|Advisors:||Garcia, Benjamin A|
|Contributors:||Molecular Biology Department|
|Publisher:||Princeton, NJ : Princeton University|
|Abstract:||ABSTRACT: The nucleosome is an octamer containing DNA wrapped around one histone H3-H4 tetramer and two histone H2A-H2B dimers, of which each histone is decorated with post-translational modifications (PTMs). Mass spectrometry (MS) is an essential tool for quantitatively studying histone PTMs and examining nucleosome content. Studies indicate that the H3-H4 tetramer is conserved during DNA replication, suggesting that "old" tetramers could serve as a template for the modification of newly synthesized tetramers. Here we present our work on a dual labeling strategy that merges bioorthogonal chemistry with mass spectrometry in an effort to better understand how PTMs are acquired on newly synthesized histones in mammalian cells. Briefly, HeLa S3 cells are dually labeled with the methionine analog azidohomoalanine (AHA) and heavy isotope labeled amino acids. Heavy amino acid labeling allows for marking of newly synthesized histones while AHA incorporation allows for their isolation using bioorthogonal chemistry. AHA containing mononuclesomes were covalently linked via a copper catalyzed reaction to a FLAG-alkyne peptide, immunoprecipitated and subjected to quantitative mass spectrometry- facilitating both the distinction of new vs. old histone and the quantification of histone PTMs. The successful isolation of newly synthesized histones using this method could be utilized to address other biological questions.|
|Alternate format:||The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog|
|Type of Material:||Academic dissertations (Ph.D.)|
|Appears in Collections:||Molecular Biology|
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