Mass Spectrometry-Based Precise Identification of Citrullinated Histones via Limited Digestion and Biotin Derivative Tag Enrichment

Histone citrullination is an essential epigenetic post-translational modification (PTM) that affects many important physiological and pathological processes, but effective tools to study histone citrullination are greatly limited due to several challenges, including the small mass shift caused by this PTM and its low abundance in biological systems. Although previous studies have reported frequent occurrences of histone citrullination, these methods failed to provide a high-throughput and site-specific strategy to detect histone citrullination. Recently, we developed a biotin thiol tag that enabled precise identification of protein citrullination coupled with mass spectrometry. However, very few histone citrullination sites were identified, likely due to the highly basic nature of these proteins. In this study, we develop a novel method utilizing limited digestion and biotin derivative tag enrichment to facilitate direct in vivo identification of citrullination sites on histones. We achieve improved coverage of histone identification via partial enzymatic digestion and lysine block by dimethylation. With biotin tag-assisted chemical derivatization and enrichment, we also achieve precise annotation of histone citrullination sites with high confidence. We further compare different fragmentation methods and find that the electron-transfer-dissociation-based approach enables the most in-depth analysis and characterization. In total, we unambiguously identify 18 unique citrullination sites on histones in human astrocytoma U87 cells, including 15 citrullinated sites being detected for the first time. Some of these citrullination sites are observed to exhibit noticeable alterations in response to DNA damage, which demonstrates the superiority of our strategy in understanding the roles of histone citrullination in critical biological processes.