Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.The field of Epigenetics has become important in drug discovery as many diseases have been linked to aberrations in chromatin and changes of histone post-translational modifications (PTMs)¹ (1, 2). The core histones (H2A, H2B, H3, and H4 and their variants) undergo a multitude of PTMs. Some, like lysine acetylation, lysine mono-, di-, and trimethlyation, and serine/threonine phosphorylation are well documented, with over 100 distinct, albeit generally low abundance, modifications reported for H3 alone (3). Mass spectrometry provides an alternative to antibody-based methods for detecting and quantifying histone PTMs, as the latter are prone to problems of specificity and epitope occlusion (4, 5). The most commonly applied approach to date is known as “bottom-up” mass spectrometry and involves an initial processing of the histones into smaller peptides (6). A key development in histone PTM analysis was the deliberate chemical modification of histone tail lysines by propionic anhydride, preventing digestion of these Lys- and Arg-rich domains into peptides too short or hydrophilic to be detected in reverse-phase liquid chromatography-mass spectrometry experiments (7–9).Despite this advance, some marks like H3K4 di- and tri-methylation remain problematic; in several examples from the recent literature the H3K4me3 mark is detected either only by means of specifically targeted methods (5), with larger quantitative variation than other marks (10), or not reported among detected marks at all (3, 11–13). Alternative approaches include top-down or middle-down mass spectrometry, in which entire histones, or large segments thereof are analyzed directly (14–16), but these techniques still suffer from relatively poor sensitivity in comparison to bottom-up workflows, and must contend with the full combinatorial complexity of histone PTMs (17).The H3K4me3 mark is of low natural abundance, having a very restricted genomic localization strongly associated with active gene promotors and enhancers (18, 19), and aberrant activities of writers and erasers of that mark are associated with a variety of diseases (1, 2). Difficulties in its quantitation thus hinder the investigation of both fundamental biology and the discovery of lifesaving drugs. We therefore undertook a re-evaluation of the bottom-up histone PTM workflow, streamlining sample preparation and investigating sources of bias or sample loss. Alternatives to the standard propionylation technique were also explored, resulting in a new hybrid chemical modification workflow yielding across-the-board improvements in recovery of peptides from the N-terminal tail of histone H3, and dramatically improved detection of hydrophilic peptides with marks like H3K4me2/me3.     

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano–liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N^α-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.Histones are the most intensively studied group of basic nuclear proteins and are of great importance with regard to the organization of chromatin structure and control of gene activity. They are highly conserved during evolution, binding to and condensing eukaryotic chromosomal DNA to form chromatin. The fundamental chromatin subunit is the nucleosome, in which 166 bp of DNA are wrapped around a core histone octamer and a further ∼40 bp constitute the linker between one nucleosome core and the next. The histone octamer contains two molecules of each of the core histones H2A, H2B, H3, and H4. A fifth type of histone, referred to as linker histone (H1, H5), binds to both the DNA on the outer surface of nucleosomes and the linker DNA.There are numerous microsequence variants of linker and core histones (except H4) differing only slightly in primary sequence. In rat testis, for example, six somatic H1 subtypes, designated as H1a, H1b, H1c, H1d, H1e, and H1.0, as well as germ cell specific subtypes (i.e. H1t, H1T2, and HILS1), have been identified (1–3). Under various biological conditions, all histone proteins, for both linker and core histones, are subjected to post-translational modifications, including phosphorylation, acetylation, methylation, ubiquitination, deamidation, glycosylation, and ADP-ribosylation, which have a great influence on the epigenetic control of gene expression (4–6). The multitude of histone proteins resulting from closely related sequence variants and post-translational modifications, as well as their highly basic nature combined with hydrophobic properties, provides a major analytical challenge in current proteomics research. Over the past several years, considerable efforts have been expended to develop methods to identify the specific sites of histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the dominant technique for their characterization (7–14). However, because histone proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still problematic, as digestion with many commonly used enzymes (e.g. trypsin, Lys-C, etc.) causes the formation of many short and polar peptides that poorly interact with the reverse-phase (RP)¹ material and go undetected by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). To overcome this problem, chemical derivatization such as propionylation is often applied (15, 16).Capillary electrophoresis (CE) overcomes this disadvantage; this technique allows separations based on the mass-to-charge ratio of peptides and does not utilize their hydrophobic nature as a separation principle. The methods of electrophoresis and LC and their applicability for histone analysis have been reviewed in detail by Lindner (17). CE has proven to be a remarkably powerful method for separating individual histones and their modified forms based on their different electrophoretic mobilities. Using a bare fused silica capillary and hydroxypropylmethyl cellulose (HPMC) as a buffer additive in order to avoid undesired protein adsorption, different core and linker histones and their multiply phosphorylated and acetylated forms were successfully separated via capillary zone electrophoresis (CZE) (18–22). So far, no data have been published about the identification of histone modifications by means of capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS). LC is given preference over CE because of the difficulty of achieving on-line interfacing of CE with MS that allows stable electrospray processes without compromising the quality of separation or the detection sensitivity. However, CE-MS is a promising technique with constantly increasing importance, as documented by numerous articles (23–26).Various interfaces have been constructed to improve CESI-MS coupling (27, 28). Sheathflow interfaces are the most widely used, and although the drawback of having to dilute the analyte is inherent in this kind of interface, they offer stable electrophoretic separations and allow greater versatility in the choice of background electrolyte (BGE) and the range of flow rates (29–32). Sheathless interfaces have generated interest because no sheath liquid is added, which leads to enhanced detection sensitivity (33, 34). However, they have not been used frequently because of their limited robustness and lack of well-established interfaces and routine analysis protocols. The most widely used method for establishing the terminating electrical contact is coating the outer surface of the CE capillary tip with a conductive material (35–37). Unfortunately, the lifetimes of such coatings are generally very limited, as they suffer from deterioration under the influence of the high voltages applied.A recently published concept of a sheathless interface based on a separation capillary with a porous tip acting as a nanospray emitter overcomes these disadvantages (38). The capillary tip is etched using hydrofluoric acid until the capillary wall becomes so thin and porous that an electric contact can be established. The performance of this methodology, which combines the low-flow characteristics of CE with an integrated ESI source, is described in Refs. 39–41. Applications such as the analysis of intact proteins (42), protein–protein and protein–metal complexes (43), and ribosomal protein digests from E. coli (44) have been published. Method-inherent advantages of CESI-MS are highly efficient separations, low flow rates leading to reduced ion suppression, and greater sensitivity (40). In contrast to nano-LC, no column equilibration is needed, there are no gradient effects, and the instrumentation is less maintenance-intensive.Our group recently described important features of CESI-MS and reported the comparison of this method with LC-ESI-MS for the analysis of a 5% perchloric acid extraction of rat testis consisting mainly of different histone H1 subtypes (39). The performance of both techniques was evaluated regarding analysis time, protein sequence coverage, and number and molecular mass distribution of the identified peptides. The CESI-MS method provided shorter analysis times, narrower peaks yielding high signals, and the identification of a greater number of low molecular mass range peptides than LC-ESI-MS (39).In the current study, we investigated the analysis of post-translationally modified peptides, particularly phosphopeptides, obtained from endoproteinase Arg-C digested histones from rat testis; this organ contains the whole set of somatic and germ cell specific H1 histones, as well as numerous modified core histone proteins. CESI-MS and LC-ESI-MS were compared regarding the number and type of identified modified peptides. Without any pre-separation of the perchloric acid extraction, we found numerous known and novel modification sites in linker histones. In addition, immobilized metal-affinity chromatography (IMAC) experiments were utilized to enrich phosphopeptides prior to MS analysis. CESI-MS was also used for the rapid identification of post-translational modifications (PTMs) of rat testis core histones, which were pre-fractionated via RP-HPLC and digested with Arg-C. Using core histones from butyrate-treated mouse erythroleukemia cells, we further demonstrated that our method achieves excellent separations of intact histone subtypes and their multiply modified forms and enables the detection of the extent of PTMs in a fast and reproducible way. Our work represents the first detailed characterization of modified linker and core histone peptides and clearly demonstrates that CESI-MS is a promising alternative tool for epigenetic studies.