Characterization of histones and their posttranslational modifications by mass spectrometry

In this dissertation, mass spectrometry-based approaches were developed to characterize histones and their posttranslational modifications (PTMs). Histones are the primary protein component of chromatin. Histone tails are subject to a variety of PTMs including acetylation, methylation, and phosphorylation. These PTMs play critical roles in the regulation of chromatin remodeling and gene transcription. Therefore there is a need for better approaches to characterize histones and their PTMs. Yeast are chosen as the model for my studies due to many of their beneficial properties.;Methods for yeast histone preparation, separation, and LC-MS were developed and described in Chapter 2. Through extensive washing of nuclei prior to lysis and acid extraction, yeast histones were extracted from yeast whole cells with a high yield and purity. The isolated yeast histones were then separated by use of 16.5% Tris-tricine-SDS gels and characterized by LC-MS with trifluoroacetic acid (TFA) as the ion-pairing agent with minimal TFA effects. By using the methods developed in Chapter 2, histone acetylation and methylation patterns in yeast were studied (Chapter 3). We found that acetylation and trimethylation have different effects on retention time in reversed-phase liquid chromatography. The former shifts retention time, while the latter does not. By using retention time, acetylation and trimethylation were unambiguously distinguished even on a low-resolution mass spectrometer. In Chapter 4, we examined five JmjC domain-containing proteins (Ecm5, Gis1, Rph1, Jhd1, and Jhd2) in yeast for demethylase activities. Lysine methylation used to be considered irreversible. By using mass spectrometry-based approaches, we found that these proteins (except for Ecm5) displayed demethylase activity. In particular, Rph1 exhibited specificity toward H3K36 di- and trimethylation and Jhd2 had specificity toward H3K4 trimethylation. Cross-talk between methylation and acetylation was assessed in Chapter 5 by alterating the expression of the JmjC proteins. Our results show that H3 acetylation changes significantly in all strains expect for ecm5Delta. LC-MS/MS results indicate that Gis1 and Jhd1 facilitate K56Ac, while Rph1 decreases the level of K56Ac. Taking into consideration that the JmjC proteins are histone demethylases, this study implies that H3 methylation is correlated with acetylation on H2B1, H2B2, and H3.