Quantitative proteomics methods for the analysis of histone post-translational modifications

Histones are highly conserved, basic proteins found in eukaryotic cell nuclei. They organize and package DNA strands into nucleosome core particles (NCPs), the fundamental repeating units of eukaryotic chromatin. The histones are subject to a wide variety of posttranslational modifications (PTMs) including acetylation, methylation and phosphorylation. These PTMs play an essential role in DNA-replication, transcription, and chromatin assembly. Alterations in histone PTM abundances have been implicated in several types of cancer. For example, the global loss of trimethylation at H4K20 and acetylation at H4K16 is a hallmark of human cancers. Thus, characterization of histone PTMs and their dynamics is extremely useful for elucidating normal cellular functions and molecular pathways that lead to diseases.  Traditionally, histone PTMs are analyzed using antibody-based approaches such as western blot and chromatin immunoprecipitation (ChIP) assays. These methods, however, suffer from several limitations including antibody cross-reactivity, epitope occlusion, and the cost and difficulty in producing and validating antibodies. Over the last decade, mass spectrometry (MS) has emerged as a powerful technique for the characterization and quantification of histone PTMs. MS offers several advantages over the traditional approaches including reproducibility, specificity, and ability to rapidly analyze numerous PTMs in a single experiment. In this thesis, the development and applications of novel analytical tools for the identification and quantification of histone PTMs are presented.  First, a method useful for measuring the global and site specific changes in histone acetylation is described. This method combines intact mass analysis and peptide sequencing approaches to study the global and site specific changes in histone acetylation during in vitro assays with yeast Rtt109 and its chaperone (Asf1 or Vps75).  Second, a method for analysis of isomeric histone peptides is presented. This method combines a high resolution LC-MS/MS with a novel bioinformatics tool called Iso-PeptidAce to deconvolute mixed spectra of co-eluting isomeric peptides. We benchmarked Iso-PeptidAce using mixtures of synthetic isomeric peptides. We demonstrated its capability in histones isolated from human erythroleukemic (K562) cells treated with clinically relevant histone deacetylase inhibitors (HDACi) and in affinity-purified S. cerevisiae histones bound to chromatin assembly factor-1 (CAF-1). Third, by employing the above methods, an in-depth quantitative analysis of the substrate specificities of several fission yeast HATs and HDACs was assessed. We determined the acetylation site occupancy of multiple lysines in histones isolated from a control or mutant strains lacking specific HAT or HDAC activities. Our analysis identified several known and novel HAT and HDAC target sites. Our data also defined the division of labor between the different HATs and HDACs in maintaining the steady-state level of histone acetylation. Overall, we anticipate that the methods described in this thesis will address some of the existing challenges facing the chromatin field. Moreover, the data presented will provide valuable insights for future genetic and biochemical studies involving the fission yeast.

Keyphrases: Histones, Mass spectrometry, Histones, Post-translational modifications, Isomeric peptides, Histones acetyltransferases, Histone deacetylases