Histone post-translational modifications (PTMs) are critical epigenetic regulatory factors. Histone PTMs are highly dynamic and complicated, encompassing over 30 structurally diverse modifications across nearly 180 amino acid residues, which generated extensive information regarding histone marks. In proteomics-based characterization of histone PTMs, chemical derivatization and antibody-based affinity enrichment were frequently utilized to improve the identification depth. However, chemical derivatization suffered from the occurrence of side reactions, and antibody-based affinity enrichment focused on specific PTM types of interest. In this research, we developed a multi-step fractionation strategy for comprehensively unbiased detection of histone PTM sites. By combining protein-level fractionation with peptide-level alkaline and acid phase fractionation, we developed the Multidimensional Fractionation based Histone Mark Identification Technology (MudFIT) and increased PTM identification to a total of 264 histone PTM sites. To the best of our knowledge, this strategy achieved the most comprehensive characterization of histone PTM sites in a single proteomics study. Using the same starting amount of sample, MudFIT identified more Kac sites and Kac peptides than those in antibody-based acetylated peptide enrichment. Moreover, in addition to well-studied histone marks, we discovered 36 potential new histone PTM sites including H2BK116bu, H4R45me2, H1K63pr, and uncovered unknown histone PTM types like aminoadipic on lysine and nitrosylation on tyrosine. Our data provided a method and resource for in-depth characterization of histone PTM sites, facilitating further biological understanding of histone marks.
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