Characterization of histones and their post-translational modifications using reversed-phase high performance liquid chromatography and mass spectrometry

The current research focuses on the characterization and quantitation of histone variants and their post-translationally modified isoforms using a series of mass spectrometry based technologies. An LC-MS method was optimized to facilitate the characterization of linker and core histones. A novel 2D LC-MS (HAPR LC-MS) was further developed to characterize not only histones but also non-histone chromatin-associated proteins (NHCPs) that are involved in a variety of genetic activities and generally function together with histones during transcription activation or repression. The optimized LC-MS and HARP LC-MS methods were then successfully applied to investigating histories from different sources including the blood of CLL patients, yeast S. cerevisiae and human cells. AU-PAGE, SDS-PAGE, peptide mass mapping and tandem mass spectrometry were also employed to investigate sequence variants and their posttranslational modifications. Two histone H2A variants (H2AFL/G and H2AFM*) were found to decrease in abundance in the CLL patient as compare to the normal individual, which suggests these variants may potentially be biomarkers for prognosis of CLL in the early stage. Using recombinant yeast strains, K91 of histone H4 was indicated to regulate H4 acetylation and K79 to play crucial roles in the expression of core histones, especially H2B ( HTB2), H4 and H3. The acetylation pattern of H4 from the wild type yeast was established to be similar to that of mammals: initial K16(Ac) followed by K12(Ac) and K8(Ac) or K5(Ac). Characterization of post-translational modifications was also achieved for the minor histone variant H2A.Z and its associated H2B in yeast. Novel modifications were discovered for these species. Ubiquitination of H2A and H2B were also investigated and a novel ubiquitinated site was found on K108 of H2B in addition to the known sites of H2A K119-Ub and H2B K120-Ub. Finally, a quantitative and dynamic study of histone H4 acetylation by depsipeptide, a histone deacetylase inhibitor and potential anticancer agent, was performed.