A two-pronged approach using specialized peptide detection and clustering tools was developed to profile changes in histone post-translational modifications (PTMs). The extent and nature of modification was inferred by comparing the mass profiles of intact core histones from nano LC-MS experiments. Histones displaying changes in their intact mass profiles were fractionated, derivatized with propionic anhydride, and digested with trypsin prior to nano LC-MS analyses. Our methodology was validated by comparing the abundance of histone PTMs in wild type and mutant strains of Saccharomyces cerevisiae lacking the histone acetyltransferase Rtt109 and a nucleosome assembly factor known as Asf1. Both Rtt109 and Asf1 were previously found to be essential for acetylation of histone H3 lysine 56 (H3K56ac), a modification that plays an important role in the response to genotoxic agents that interfere with DNA replication. The generation of ion abundance distribution plots enabled a rapid and comprehensive profiling of histone peptides. Our analytical methodology and data mining approach showed that most common histone PTMs were unaffected in mutant yeast cells lacking Rtt109 and Asf1. However, a subpopulation representing 17% of all H3 histones in wild type cells showed an acetylated K56 residue that was significantly reduced in both mutant strains. Our generic strategy for histone PTM profiling can be applied to assess global changes in histone PTMs across sample sets and to establish structure-function relationships.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|