In eukaryotes, the fundamental unit of chromatin is nucleosome, which contains approximately 147 base pairs of DNA wrapped around an octamer of core histones (two each of histones H2A, H2B, H3, and H4), along with a linker histone H1 [1]. Histones undergo both enzymatic and non-enzymatic post-translational modifications (PTMs), with over 30 structurally diverse PTM types at approximately 180 amino acid residues [2]. These modifications regulate gene expression, DNA replication, and cell cycle progression through the following mechanisms: 1) They modulate chromatin conformation by altering charge distribution. 2) They recruit downstream effectors, such as transcription factors, enzymes associated with DNA-histone interactions, chromatin remodelers, and histone chaperones [3]. Dysregulation of crucial histone PTMs is involved in various diseases, including neurological disorders, inflammation, and cancers [4], posing them as potential therapeutical targets.

Histone PTMs are highly dynamic, reversible, conserved and complicated. They are regulated by writers (enzymes catalyzing modification addition), erasers (enzymes mediating modification removal), and readers (proteins recognizing specific structure) [5]. Histone PTMs detection commonly relied on western blot (WB) and chromatin immunoprecipitation sequencing (ChIP-seq) [6]. WB utilizes antibodies to detect PTMs with high specificity and affinity, and has been a crucial tool for epigenetics research. However, antibodies show several limitations, such as epitope occlusion, cross-reactivity, and uncertain binding efficiency [7]. ChIP-seq integrates chemical cross-linking, affinity enrichment, and high-throughput sequencing to define the interaction of histones with specific chromosomal sites in vivo. Cleavage under targets and release using nuclease (CUT&RUN) [8] and cleavage under targets and tagmentation (CUT&Tag) [9] have been further developed to streamline the experimental procedures, reduce sample input amounts, and shorten detection cycles. However, these methods still rely on antibody and are limited by low-throughput. Even the multiplexed quantitative chromatin immunoprecipitation sequencing (MINUTE-ChIP) approach can only simultaneously analyze up to 6 modification sites [10], restricting comprehensive histone code analysis.

Since the 1880s, mass spectrometry (MS) has emerged as a powerful technique for the determination of the amino acid sequences of proteins [11,12]. It is an analytical technique that determines the mass-to-charge (m/z) ratio of ions in the gas phase for molecules constituting a sample. Over the past two decades, substantial technological improvements in the accuracy and resolution of MS have made it the predominant approach for histone PTM identification within complex protein samples [13]. MS-based proteomics contains three primary analysis strategies: top-down, middle-down, and bottom-up. The top-down approach analyzes intact histone proteins without proteolytic digestion, offering more comprehensive information about coexisting PTMs compared to the other two approaches. However, the co-elution and co-fragmentation of multiple isoforms may reduce detection sensitivity. The middle-down strategy utilizes proteases such as Glu-C or Asp-N to analyze relatively long polypeptides, providing robustness and reliability in characterizing co-existing PTMs. Nevertheless, its application in chromatin-related studies remains rare, likely due to the high complexity of the methodology and data analysis [14]. In a typical bottom-up experiment, histones are enzymatically digested with trypsin. With the high throughput and sensitivity, the bottom-up strategy has been widely adopted for the robust and accurate characterization of histone PTMs [1615 16]. However, this approach often generates peptides that are too short to be effectively retained on reversed-phase chromatography columns [17]. The integration of MS-based bottom-up method and quantitative strategies, such as metabolic labeling, chemical labeling [18], and label-free [19], facilitates systematic PTM quantification. Various search engines have been developed to determine optimal matching peptide ion identifications. Three primary categories of search engines include: 1) Sequence search engines (e.g., Mascot, Sequest, X!Tandem, Sonar, ProbID, Andromeda, and pfind), which typically align acquired spectra against theoretical spectra generated from the possible peptide sequences within a provided protein sequence database [20]. These engines are most popularly used for histone PTM characterization. 2) Spectral library search engines (e.g., SpectraST, X!Hunter, and Bibliospec), which compare spectra against a library of previously identified spectra [21]. 3) De novo search engines (e.g., PEAKS and PepNovo), which infer peptide sequences directly from the spectral peak patterns, independent of reference sequences or prior spectrum libraries [22]. This review focuses on the advancements in MS-based proteomic technologies for histone PTM identification (Supplementary Table 1), with an emphasis on the latest analytical strategies and the discovery of novel histone PTMs.