Progress in epigenetic histone modification analysis by mass spectrometry for clinical investigations

Abstract

Chromatin biology and epigenetics are scientific fields that are rapid expanding due to their fundamental role in understanding cell development, heritable characters and progression of diseases. Histone post-translational modifications (PTMs) are major regulators of the epigenetic machinery due to their ability to modulate gene expression, DNA repair and chromosome condensation. Large-scale strategies based on mass spectrometry have been impressively improved in the last decade, so that global changes of histone PTM abundances are quantifiable with nearly routine proteomics analyses and it is now possible to determine combinatorial patterns of modifications. Presented here is an overview of the most utilized and newly developed proteomics strategies for histone PTM characterization and a number of case studies where epigenetic mechanisms have been comprehensively characterized. Moreover, a number of current epigenetic therapies are illustrated, with an emphasis on cancer.

Keywords: cancer; chromatin; epigenetics; histone; histone variants; mass spectrometry; post-translational modifications; proteomics; translational medicine.

Figure 1. The genome and the phenotype during development and role of epigenetics

Representation of the massive change of a human phenotype during development, while maintains a nearly identical DNA sequence through the entire lifecycle. Epigenetics is a major effector in this process, as gene expression is modified and then modifications inherited during embryonic development of specialized tissues.

Figure 2. Antibody steric hindrance during histone post-translational modification recognition

Schematic representation of an antibody that is (A.) targeting its epitope without any interference, (B.) recognizing `off-target' epitope, (C.) not recognizing the epitope due to a nearby secondary histone modification and (D.) not recognizing the epitope due to a secondary histone modification distantly located from the target.

Figure 3

Summary of bottom-up, top-down and middle down approaches in histone post-translational modification analysis

Figure 4. The mass spectrometry-based proteomics strategies for histone analysis

(A.) Histones are digested into short peptides when adopting the bottom-up strategy (top left). The use of short peptides prevents the acquisition of combinatorial post-translational modification (PTM) data, as the peptides detected cannot be used to verify whether two modifications (e.g. red and yellow circles) were present simultaneously on the same histone protein. The middle-down strategy partially overcomes to this issue, since it allows for the analysis of the entire histone tail (top right), but it cannot provide data of histone PTMs localized on the nucleosome core. Top-down allows for the analysis of the intact histone (bottom left). The ultimate goal would be the analysis of the entire nucleosome (most probably cross-linked), but mass spectrometry of histone complexes, especially if modified, is still highly challenging (bottom right). (B.) Example of the increasing complexity in going from bottom-up to top-down analysis of histone H3 (green colored and highlighted by a pink glow). For example considering only methylation, a histone H3 peptide has on average 2–3 modifiable residues, i.e. arginine and lysine residues. An average bottom-up peptide with two lysine and one arginine residue could exist in 64 different combinations (K can be unmodified, me1, me2 and me3; R can be unmodified, me1, me2). Of these, 54 have at least one isobaric form, defined as alternative PTM combination with the same intact mass (e.g. H3K27me2K36me1 vs H3K27me2K36me2). In the middle-down and the top-down strategy this issue grows exponentially, as the only species with no isobaric forms are the completely unmodified and the completely methylated sequence. Even though it is less likely that all the possible combinations are exist in nature, this issue is challenging as it leads to highly complex MS/MS spectra, due to the myriad of co-fragmenting proteoforms.

Figure 5. Ion map of a typical bottom-up and middle-down LC-MS run

(A.) Bottom-up LC-MS ion map of histone extract. A typical bottom-up analysis is performed with a relatively short gradient (< 1 hour) using C₁₈ chromatography, and histone peptides are eluted in sharp peaks (<1 min) with a precise but disperse order. Different peptide sequences and with multiple modifications are spread all over the chromatogram. However, some trends are conserved, and can be used to confidently identify histone peaks. For instance, a peptide carrying only one modification and derivatized with propionic anhydride is eluted with the order di- and trimethylated, acetylated, unmodified (propionylated) and monomethylated (propionylated). (B.) Middle-down LC-MS ion map of histone H3 N-terminal tails. A typical middle-down analysis is performed using a long gradient (2–3 hours) with WCX-HILIC chromatography. Histone variants are usually fractionated in different tubes, in order to achieve a more in depth characterization of a given variant. Histone tails elution has a specific trend, where more modified species are eluted first. Acetylation (ac) has a larger influence in the retention time as compared to methylation (me).

Figure 6. Deconvoluted MS/MS spectrum of a modified histone N-terminal tail

MS/MS spectra generated using the middle-down strategy are usually rich in heavily charged product ions, that are deconvoluted into single charges to assist database searching and identification. The present spectrum was obtained using ETD fragmentation, which generates c and z ions, and split into three rows for simpler visualization.