Improved Dynamic Range, Quantitation, and Characterization of Histone H4 Post-Translational Modifications: A Top Down Mass Spectrometric Approach
Pesavento, James J.
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https://hdl.handle.net/2142/85454
Description
Title
Improved Dynamic Range, Quantitation, and Characterization of Histone H4 Post-Translational Modifications: A Top Down Mass Spectrometric Approach
Author(s)
Pesavento, James J.
Issue Date
2006
Doctoral Committee Chair(s)
Kelleher, Neil L.
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Cell
Language
eng
Abstract
"Intimately associated with DNA, histone proteins serve as both a structural scaffold for DNA packaging into the nucleus and an epigenetic means for the regulation of gene expression. One such histone-based mechanism for transcriptional regulation is post-translational modification (PTM) of histones H2A, H2B, H3 and H4. Combinations of modifications such as acetylation, methylation, and phosphorylation have been hypothesized to create a ""histone code"" that influences gene transcription, gene silencing, and chromatin formation. Essential for complete understanding of this code is an efficient methodology for detection, exact localization and quantitation of combinations of modifications at specific sites. We combine here gas-phase concentration and purification of human histone H4 inside a Quadrupole-Fourier Transform Mass Spectrometer hybrid (Q-FTMS) with Top Down fragmentation using Electron Capture Dissociation (ECD). We extend the use of Top Down MS to assess how the abundance of each modified histone H4 form changes in synchronized cells progressing through the cell cycle in order to identify PTMs and combinations of PTMs that are associated with cell cycle specific events such as replication and mitosis. The many observed combinations of modifications on H4 led us to develop a novel database searching strategy to simplify data analysis. Histone H4 was ""shotgun annotated"", resulting in the population of a database with masses of hypothetical modified H4 forms. Querying this database with ECD spectra rich in fragment-ions, we found that this approach quickly finds the correct modified H4 form. We also developed an additional chromatographic approach that increased our dynamic range from 102 to >10 4, allowing the characterization and quantitation of >35 chemically distinct forms of H4 in HeLa cells, many of which have not been described previously. During the quantitation of these 39 distinct forms, we developed methods that dealt with challenges associated with intact proteins ( i.e., partial oxidation of histones) and dissected isomeric mixtures of H4 PTMs). The prevalence of multiply modified H4 revealed here suggests that current views of histone PTM function are biased by the limited ability of other approaches to account for combinatorial modification."
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