Two-dimensional liquid chromatography system for online top-down mass spectrometry

An online metal-free weak cation exchange-hydrophilic interaction LC/RPLC system has been developed for sensitive, high-throughput top-down MS. Here, we report results for analyzing PTMs of core histones, with a focus on histone H4, using this system. With just ～24?μg on-column of core histones (H4, H2B, H2A, and H3) purified from human fibroblasts, 41 H4 isoforms were identified, with the type and location of PTMs unambiguously mapped for 20 of these variants. Compared to corresponding offline studies reported previously, the online weak cation exchange-hydrophilic interaction LC/RPLC platform offers significant improvement in sensitivity, with several orders of magnitude reduction in sample requirements and a reduction in the overall analysis time. To the best of our knowledge, this study represents the first online 2-D LC-MS/MS characterization of core histone mixture at the intact protein level.     

Liquid chromatography mass spectrometry profiling of histones

Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RPLC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS.     

Middle-down proteomics: a still unexploited resource for chromatin biology

Introduction: Analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has become a fundamental tool for the characterization of chromatin composition and dynamics. Histone PTMs benchmark several biological states of chromatin, including regions of active enhancers, active/repressed gene promoters and damaged DNA. These complex regulatory mechanisms are often defined by combinatorial histone PTMs; for instance, active enhancers are commonly occupied by both marks H3K4me1 and H3K27ac. The traditional bottom-up MS strategy identifies and quantifies short (aa 4–20) tryptic peptides, and it is thus not suitable for the characterization of combinatorial PTMs.

Areas covered: Here, we review the advancement of the middle-down MS strategy applied to histones, which consists in the analysis of intact histone N-terminal tails (aa 50–60). Middle-down MS has reached sufficient robustness and reliability, and it is far less technically challenging than PTM quantification on intact histones (top-down). However, the very few chromatin biology studies applying middle-down MS resulting from PubMed searches indicate that it is still very scarcely exploited, potentially due to the apparent high complexity of method and analysis.

Expert commentary: We will discuss the state-of-the-art workflow and examples of existing studies, aiming to highlight its potential and feasibility for studies of cell biologists interested in chromatin and epigenetics.     

Properly reading the histone code by MS‐based proteomics

Histone proteins are essential elements for DNA packaging. Their PTMs contribute in modeling chromatin structure and recruiting enzymes involved in gene regulation, DNA repair, and chromosome condensation. This fundamental aspect, together with the fact that histone PTMs can be epigenetically inherited through cell generations, enlightens their importance in chromatin biology, and the consequent necessity of having biochemical techniques for their characterization. Nanoflow LC coupled to MS (nanoLC‐MS) is the strategy of choice for protein PTM accurate quantification. However, histones require adjustments to the digestion protocol such as lysine derivatization to obtain suitable peptides for the analysis. nanoLC‐MS has numerous advantages, spanning from high confidence identification to possibility of high throughput analyses, but the peculiarity of the histone preparation protocol requires continuous monitoring with the most modern available technologies to question its reliability. The work of Meert et al. (Proteomics 2015, 15, 2966–2971) establishes which protocols lead to either incomplete derivatization or derivatization of undesired amino acid residues using a combination of high resolution MS and bioinformatics tools for the alignment and the characterization of nanoLC‐MS runs. As well, they identify a number of side reactions that could be potentially misinterpreted as biological PTMs.     

Tackling aspecific side reactions during histone propionylation: The promise of reversing overpropionylation

Histone proteins are essential elements for DNA packaging. Moreover, the PTMs that are extremely abundant on these proteins, contribute in modeling chromatin structure and recruiting enzymes involved in gene regulation, DNA repair and chromosome condensation. This fundamental aspect, together with the epigenetic inheritance of histone PTMs, underlines the importance of having biochemical techniques for their characterization. Over the past two decades, significant improvements in mass accuracy and resolution of mass spectrometers have made LC‐coupled MS the strategy of choice for accurate identification and quantification of protein PTMs. Nevertheless, in previous work we disclosed the limitations and biases of the most widely adopted sample preparation protocols for histone propionylation, required prior to bottom‐up MS analysis. In this work, however, we put forward a new specific and efficient propionylation strategy by means of propionic anhydride. In this method, aspecific overpropionylation at serine (S), threonine (T) and tyrosine (Y) is reversed by adding hydroxylamine (HA). We recommend using this method for future analysis of histones through bottom‐up MS.     

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.     

Histone modification in Drosophila

Post-translational modifications (PTMs) of nucleosomal core histones play roles in basic biological processes via altering chromatin structure and creating target sites for proteins acting on chromatin. Several features make Drosophila a uniquely effective model for studying PTMs. Position effect variegation, polycomb repression, dosage compensation and several other processes extensively studied by the powerful tools of Drosophila genetics as well as polytene chromosome cytology reveal information on the dynamic changes of histone PTMs and factors that deposit, remove and recognize these. Recent determination of the genome-wide distribution of more than 20 different histone PTM types has resulted in a highly detailed view of chromatin landscape. This review samples from the wealth of data these analyses have provided together with data resulting from gene-targeted studies on the distribution and role of specific histone modifications and modifiers. As an example of the complex interactions among PTMs, we will also discuss crosstalk involving specific phosphorylated and acetylated histone forms.     

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.     

“Direct” and “Indirect” Effects of Histone Modifications: Modulation of Sterical Bulk as a Novel Source of Functionality

The chromatin-regulatory principles of histone post-translational modifications (PTMs) are discussed with a focus on the potential alterations in chromatin functional state due to steric and mechanical constraints imposed by bulky histone modifications such as ubiquitin and SUMO. In the classical view, PTMs operate as recruitment platforms for histone “readers,” and as determinants of chromatin array compaction. Alterations of histone charges by “small” chemical modifications (e.g., acetylation, phosphorylation) could regulate nucleosome spontaneous dynamics without globally affecting nucleosome structure. These fluctuations in nucleosome wrapping can be exploited by chromatin-processing machinery. In contrast, ubiquitin and SUMO are comparable in size to histones, and it seems logical that these PTMs could conflict with canonical nucleosome organization. An experimentally testable hypothesis that by adding sterical bulk these PTMs can robustly alter nucleosome primary structure is proposed. The model presented here stresses the diversity of mechanisms by which histone PTMs regulate chromatin dynamics, primary structure and, hence, functionality.     

Validation of an LC‐MS based approach for profiling histones in chronic lymphocytic leukemia

The in vitro evaluation of histones and their PTMs has drawn substantial interest in the development of epigenetic therapies. The differential expression of histone isoforms may serve as a potential marker in the classification of diseases affected by chromatin abnormalities. In this study, protein profiling by LC and MS was used to explore differences in histone composition in primary chronic lymphocytic leukemia (CLL) cells. Extensive method validations were performed to determine the experimental variances that would impact histone relative abundance. The resulting data demonstrated that the proposed methodology was suitable for the analysis of histone profiles. In 4 normal individuals and 40 CLL patients, a significant decrease in the relative abundance of histone H2A variants (H2AFL and H2AFA/M*) was observed in primary CLL cells as compared to normal B cells. Protein identities were determined using high mass accuracy MS and shotgun proteomics.     

Enrichment and characterization of histones by two-dimensional hydroxyapatite/reversed-phase liquid chromatography-mass spectrometry

Here we report a novel two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method that combines offline hydroxyapatite (HA) chromatography with online reversed-phase liquid chromatography-mass spectrometry (HA/RP LC-MS). The 2D LC-MS method was used to enrich and characterize histones and their posttranslational modifications. The 2D HA/RP LC-MS approach separates histones based on their relative binding affinity to DNA and relative hydrophobicity. HA/RP separations showed improvement in the number of histone isoforms detected as compared with one-dimensional RP LC-MS of acid-extracted histones. The improved histone fractionation resulted in better detection of lower abundant histone variants as well as their posttranslationally modified isoforms. Histones eluted from the HA/RP in the following order: H1, H2A/H2B heterodimers followed by H3/H4 heterotetramers, as predicted from their spatial organization in nucleosomes for binding affinity to DNA. Comparison between HA-purified and acid-extracted histones revealed similar histone profiles with the exception that the HA fractions showed a greater number of H1 isoforms. Two elution conditions were also examined: batch elution and salt gradient elution. Although both elution techniques were able to fractionate the histones sufficiently, the salt gradient approach has the most potential for purification of selected histone isoforms.     

Identification of lysine 37 of histone H2B as a novel site of methylation

Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function.     

Quantitative assessment of chemical artefacts produced by propionylation of histones prior to mass spectrometry analysis

Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot‐gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain “Arg‐C like” digestion products that are more suitable for LC‐MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. 1 is a quantitative assessment of methyl‐esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059–2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl‐esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl‐esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation.     

Middle‐Down MS is Ready to Answer Complex Questions in Chromatin Biology

Histones are the most abundant protein family in the cells of complex organisms such as mammals and, together with DNA, they define the backbone of chromatin. Histone PTMs are key players of chromatin biology, as they function as anchors for proteins that bind and modulate chromatin readout, including gene expression. Middle‐down mass spectrometry (MS) has been optimized for about 10 years to study histone N‐terminal tails, but it has been rarely applied to identify the role of coexisting histone marks in biology. In this work, Jiang et al. used middle‐down MS to study the dynamics of coexisting PTMs on histone H4 in two breast cancer cell lines. 1 They found that overall serine 1 phosphorylation (S1ph) is mildly regulated during the cell cycle, but S1ph coexistence frequency with acetylations and methylations on the lysine residues of the N‐terminal tail is remarkably tuned during S phase and G2/M phase. Together, the team placed another benchmark proving that MS analysis of combinatorial histone PTMs provides a more comprehensive view on chromatin state than studying individual marks. We should then constantly question ourselves regarding the limitations of analyzing single PTMs when we attempt to define their effect on protein functions.     

Global mapping of post-translational modifications on histone H3 variants in mouse testes

Mass spectrometry (MS)-based characterization is important in proteomic research for verification of structural features and functional understanding of gene expression. Post-translational modifications (PTMs) such as methylation and acetylation have been reported to be associated with chromatin remodeling during spermatogenesis. Although antibody- and MS-based approaches have been applied for characterization of PTMs on H3 variants during spermatogenesis, variant-specific PTMs are still underexplored. We identified several lysine modifications in H3 variants, including testis-specific histone H3 (H3t), through their successful separation with MS-based strategy, based on differences in masses, retention times, and presence of immonium ions. Besides methylation and acetylation, we detected formylation as a novel PTM on H3 variants in mouse testes. These patterns were also observed in H3t. Our data provide high-throughput structural information about PTMs on H3 variants in mouse testes and show possible applications of this strategy in future proteomic studies on histone PTMs.