To get an insight into the mechanisms of gene expression regulation in eukaryotic organisms, it is necessary to decipher the connection between the different chemical modifications occurring on the chromatin, at both the DNA and the associated histone proteins. Histones are basic proteins, which pack the DNA into nucleosomes, and are hot spots for several posttranslational modifications. Elucidating combinatorial histone modifications co-occurring on the same histone protein will greatly contribute to our understanding of the mechanisms involved in the development of eukaryotes. The advancements in mass spectrometry technologies, including sensitivity, accuracy, and ionization strategies, have significantly contributed to the identification of novel single and combinatorial modifications on histones isolated from model organisms. In this chapter, we describe detailed protocols applied for the extraction, purification, and processing of histones for subsequent analysis by tandem mass spectrometry, using Brassica oleracea (cauliflower), a close relative of Arabidopsis thaliana.
Keywords: Arabidopsis thaliana; Cauliflower; Chromatin; Epigenetics; Histone posttranslational modification; RP-HPLC; Tandem mass spectrometry; Trypsin digestion.