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

Abstract

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.

Figure 1.

(a) Schematic representation of optimized digestion procedures for improved histone sequencing. (i) Traditional digestion method with trypsin or LysC, (ii) Dimethyl labeling method, (iii) Partial limited enzymatic digestion. (b) The bar graph shows the histone H3.1 sequence coverage by trypsin (orange), LysC (blue), dimethyl labeling (yellow) and partial limited digestion (green).

Figure 2.. Proof-of-principle study on PAD-treated human recombinant histone H3.1 using three different enzymatic digestion methods.

Citrullination identification of PAD-treated histone H3.1 with LysC (a), trypsin (b), or partial limited digestion (c). Pink bar represents the citrullinated peptide that was identified, and blue mark refers to the arginine that was converted to citrulline. (d, e) Digested citrullinated peptide length distribution. Histogram indicates the numbers of citrullinated peptides corresponding to histone H3.1 detected by the three approaches. The color shown in the pie graph represents the peptide length (amino acid numbers). (f) Venn diagram of the number of identified citrullinated peptides in the three methods.

Figure 3.

(a) Experimental workflow of histone citrullination analysis using human astrocytoma U87 cells (biological triplicates). Cells were subjected to histone extraction, precipitation, and partial limited digestion. Citrullinated peptides were then derivatized with biotin thiol tag. Target molecules were enriched using streptavidin agarose and eluted for LC-MS/MS analysis. (b) High resolution HCD tandem MS spectra of two identified peptides with the same citrullination site in Histone 2A. The left MS spectrum (parent ion, m/z@683.34, charge +3 HCD@30) was acquired from normal trypsin/LysC digestion compared with the partial limited digestion MS spectrum on the right (parent ion, m/z@820.05, charge +3 HCD@30).

Figure 4.. Identification of histone citrullination marks.

(a) Schematic representation of core histones and linker histone H1. The histone citrullination sites uncovered in this study are indicated. Citrulline residues are marked in red. (b) Citrullination sites were identified after H₂O₂–induced DNA damage. The citrullination sites are shown in blue.