The top‐down, middle‐down, and bottom‐up mass spectrometry approaches for characterization of histone variants and their post‐translational modifications

Abstract

Epigenetic regulation of gene expression is, at least in part, mediated by histone modifications. PTMs of histones change chromatin structure and regulate gene transcription, DNA damage repair, and DNA replication. Thus, studying histone variants and their modifications not only elucidates their functional mechanisms in chromatin regulation, but also provides insights into phenotypes and diseases. A challenge in this field is to determine the best approach(es) to identify histone variants and their PTMs using a robust high-throughput analysis. The large number of histone variants and the enormous diversity that can be generated through combinatorial modifications, also known as histone code, makes identification of histone PTMs a laborious task. MS has been proven to be a powerful tool in this regard. Here, we focus on bottom-up, middle-down, and top-down MS approaches, including CID and electron-capture dissociation/electron-transfer dissociation based techniques for characterization of histones and their PTMs. In addition, we discuss advances in chromatographic separation that take advantage of the chemical properties of the specific histone modifications. This review is also unique in its discussion of current bioinformatic strategies for comprehensive histone code analysis.

Proteomic characterization of histone variants in the mouse testis by mass spectrometry-based top-down analysis

“…For example, H3t shows only three different amino acids in its sequence in comparison with the canonical H3 (15). The MS-based top-down approach is suitable for comprehensive characterization of intact histones because the proteins' sequences and their PTMs are conserved (8)(9)(10)(11)19). Previous research explored the use of proteomic analysis techniques and MS to identify the mass of TH2B (4,6,7); however, the results were not sufficiently comprehensive enough to verify MS-based characterization of testis-specific histones.…”

“…However, the digestion processes associated with active enzymes hinder extensive characterization of intact proteins, resulting in the loss of significant information pertaining to the sequencing of histone proteins due to the availability of only extremely short peptide sequences. For this reason, a top-down technique that enables characterization of intact proteins is used (8)(9)(10)(11). LC in top-down strategies is generally utilized for the separation of intact proteins using reversed phase (RP) columns (C4, C8, and C18).…”

Abundant Lysine Methylation and N-Terminal Acetylation in Sulfolobus islandicus Revealed by Bottom-Up and Top-Down Proteomics

“…This makes top-down proteomics a complementary approach to bottom-up proteomics, which offers a tremendous depth of analysis, but lacks information on intact proteoforms. Top-down proteomics has been used to characterize heavily modified histone proteoforms (23). In this study we show that it can be also successfully applied to study post-translationally modified archaeal chromatin proteins, which have comparable molecular weights (6)(7)(8)(9)(10)(11).…”

Middle-down electron capture dissociation and electron transfer dissociation for histone analysis

“…Mass spectrometry (MS) and particularly middle-down approaches are playing an increasingly important role in characterizing PTMs on histones (Cannon et al 2010;Garcia 2010;Kalli et al 2013;Wu et al 2005;Moradian et al 2014). In contrast to a typical bottom-up approach, where proteins are digested with trypsin to generate peptides generally smaller than 2500 Da, middle-down Fig.…”