A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

Differentially Isotope‐Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time‐Resolved NMR Spectroscopy

Post‐translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core histones, the establishment of different PTMs on individual “sister” histones in the same nucleosomal context, that is, asymmetric histone PTMs, are difficult to analyze. Here, we generated differentially isotope‐labeled nucleosomes to study asymmetric histone modification crosstalk by time‐resolved NMR spectroscopy. Specifically, we present mechanistic insights into nucleosomal histone H3 modification reactions in cis and in trans, that is, within individual H3 copies or between them. We validated our approach by using the H3S10phK14ac crosstalk mechanism, which is mediated by the Gcn5 acetyltransferase. Moreover, phosphorylation assays on methylated substrates showed that, under certain conditions, Haspin kinase is able to produce nucleosomes decorated asymmetrically with two distinct types of PTMs.     

基于ProteinGoggle 2.0的组蛋白H4蛋白质变体的自上而下表征

由于大量可能蛋白质变体以及每一个翻译后修饰大量可能位点的存在,核心组蛋白上密集的组合式翻译后修饰的自上而下表征一直是一个巨大的分析挑战。结合高分辨串级质谱,基于同位素质荷比和轮廓指纹比对的整体蛋白质数据库搜索引擎ProteinGoggle 2.0在组蛋白翻译后修饰的自上而下鉴定方面拥有诸多独特的优势。该文报道ProteinGoggle 2.0对HeLa核心组蛋白H4的数据库搜索及蛋白质变体的鉴定结果。基于从UniProt网站下载的人类核心组蛋白H4的纯文本文件和“鸟枪法”注释,ProteinGoggle 2.0首先创建包含所有可能蛋白质变体的理论数据库;从纯文本文件中提取的信息主要是氨基酸序列、可能的翻译后修饰（单甲基化、二甲基化、三甲基化、乙酰化和磷酸化）及氨基酸变异（A77→P）。在控制质谱水平假阳性率低于1%的前提下,共鉴定到426个蛋白质变体,这是目前为止H4蛋白质变体的最全报道。这些ProteinGoggle 2.0鉴定到的H4蛋白质变体也与之前报道的ProSightPC 2.0的鉴定结果进行了肩并肩比较。总而言之,ProteinGoggle 2.0可以对具有复杂组合修饰及氨基酸变异的蛋白质组进行数据库搜索和蛋白质变体鉴定。     

Characterization of neurohistone variants and post-translational modifications by electron capture dissociation mass spectrometry

Post-translational modifications (PTMs) of histones are intimately involved in chromatin structure and thus have roles in cellular processes through their impact on gene activation or repression. At the forefront in histone PTM analysis are mass spectrometry-based techniques, which have capabilities to produce improved views of processes affected by chromatin remodeling via histone modifications. In this report, we take the first mass spectrometric look at histone variant expression and post-translational modifications from histones isolated from rat brain tissue. Analyses of whole rat brain identified specific histone H2A and H2B gene family members and several H4 and H3 post-translational modification sites by electron capture dissociation (ECD) mass spectrometry. We subsequently compared these results to selected rat brain regions. Major differences in the expression profiles of H2A and H2B gene family members or in the post-translational modifications on histone H4 were not observed from the different brain regions using a Top Down approach. However, “Middle Down” mass spectrometry facilitating improved characterization of the histone H3 tail (1–50 residues), revealed an enrichment of trimethylation on Lys9 from cerebellum tissue compared to H3 extracted from whole brain, cerebral cortex or hypothalamus tissue. We forward this study in honor of Professor Donald F. Hunt, whose pioneering efforts in protein and PTM analyses have spawned new eras and numerous careers, many exemplified in this special issue.     

质谱技术鉴定细胞中组蛋白翻译后修饰的研究进展*

组蛋白是真核细胞中构成染色质内核小体的主要元件,其翻译后修饰蕴藏着组蛋白密码,是表观遗传学的重要内容,影响染色质的结构和功能,进而调控基因表达。组蛋白翻译后修饰形式的鉴定是揭示组蛋白密码的关键,目前质谱技术已经成为分析组蛋白及其翻译后修饰的重要工具。本文综述了组蛋白翻译后修饰鉴定方法的新进展,介绍了基于质谱技术“bottom up”和“top down”的组蛋白分析策略,及CID、ECD和ETD等鉴定组蛋白修饰位点的质谱碎片裂解技术,并结合当前研究进展,评述了质谱技术在组蛋白翻译后修饰谱的鉴定、组蛋白各种变体的测定、以及在生理过程中组蛋白修饰丰度动态变化的定量分析等方面应用的新进展。     

Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an <Emphasis Type="Italic">ADA2</Emphasis>-independent Gcn5p activity

Background  

The acetylation of the core histone NH₂-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4.     

Methyl group migration during the fragmentation of singly charged ions of trimethyllysine-containing peptides: Precaution of using MS/MS of singly charged ions for interrogating peptide methylation

Core histones are susceptible to a range of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation, and ubiquitination, which play important roles in the epigenetic control of gene expression. Here, we observed an unusual discrepancy between MALDI-MS/MS and ESI-MS/MS on the methylation of trimethyllysine-containing peptides with residues 9–17 from human histone H3 and residues 73–83 from yeast histone H3. It turned out that the discrepancy could be attributed to an unusual methyl group migration from the side chain of trimethyllysine to the C-terminal arginine residue during peptide fragmentation, and this methyl group transfer only occurred for singly charged ions, but not for doubly charged ions. The methyl group transfer argument received its support from the results on the studies of the fragmentation of the ESI- or MALDI-produced singly charged ions of several synthetic trimethyllysine-bearing peptides. The results presented in this study highlighted that caution should be exerted while MS/MS of singly charged ions is employed to interrogate the PTMs of trimethyllysine-containing peptides.     

Examining histone modification crosstalk using immobilized libraries established from ligation-ready nucleosomes

Chromatin signaling relies on a plethora of posttranslational modifications (PTM) of the histone proteins which package the long DNA molecules of our cells in reoccurring units of nucleosomes. Determining the biological function and molecular working mechanisms of different patterns of histone PTMs requires access to various chromatin substrates of defined modification status. Traditionally, these are achieved by individual reconstitution of single nucleosomes or arrays of nucleosomes in conjunction with modified histones produced by means of chemical biology. Here, we report an alternative strategy for establishing a library of differentially modified nucleosomes that bypasses the need for many individual syntheses, purification and assembly reactions by installing modified histone tails on ligation-ready, immobilized nucleosomes reconstituted in a single batch. Using the ligation-ready nucleosome strategy with sortase-mediated ligation for histone H3 and intein splicing for histone H2A, we generated libraries of up to 280 individually modified nucleosomes in 96-well plate format. Screening these libraries for the effects of patterns of PTMs onto the recruitment of a well-known chromatin factor, HP1 revealed a previously unknown long-range cross-talk between two modifications. H3S28 phosphorylation enhances recruitment of the HP1 protein to the H3K9 methylated H3-tail only in nucleosomal context. Detailed structural analysis by NMR measurements implies negative charges at position 28 to increase nucleosomal H3-tail dynamics and flexibility. Our work shows that ligation-ready nucleosomes enable unprecedented access to the ample space and complexity of histone modification patterns for the discovery and dissection of chromatin regulatory principles.

280 different patterns of histone modifications were installed in preassembled nucleosomes using PTS and SML enabling screening of readout crosstalk.     

Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid chromatography and ion trap mass spectrometry

New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 μm I.D.) and nano (100-75 μm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.     

Glycation and oxidation of histones H2B and H1: in vitro study and characterization by mass spectrometry

Among the post-translational modifications, oxidation and glycation are of special interest, especially in diseases such as diabetes, and in aging. The synergistic interaction between glycation and oxidation, also known as “glycoxidation” is highly relevant due to its involvement in the production of deleterious changes at the molecular level. Non-enzymatic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity [54]. In this report, we study glycated histones and its in vitro oxidation. Data concerning the modifications that occurred in the histones were obtained by analysis of enzymatic digests (Glu-C and Arg-C) of unmodified and glycated histones, obtained before and after oxidation. Analysis was then performed using a MALDI-MS/MS-based approach combined with nano liquid chromatography. This approach allowed us to identify histone H2B and H1 specific-sites of oxidation and to distinguish the most affected residues for each histone. The results showed the occurrence of a cumulative effect of oxidative damage in the glycated histones when subjected to in vitro oxidation, suggesting that structural changes caused by glycation induces histones to a pro-oxidant state. Comparing the data of oxidized glycated histones with data from unmodified oxidized histones, using the same model of oxidation, the results clearly show that these oxidative modifications occur earlier and more extensively in glycated histones. Furthermore, the results pointed to an increased oxidative damage in the vicinity of the glycated residues.     

Quantitative chemical proteomics approach to identify post-translational modification-mediated protein-protein interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein-protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein-protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein-protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 N-terminus (H3K4Me(3)), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me(3). This study indicates that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be used to profile protein-protein interactions mediated by PTMs, such as lysine methylation.     

Identification of ADP-ribosylated histones by the combined use of high-performance liquid chromatography and electrophoresis

Reversed-phase high-performance liquid chromatography (HPLC) was employed for analysing mono- and oligo(ADP-ribosyl)ated histones. Under the chromatographic conditions described, the ADP-ribosylated histones showed similar retention times to the unmodified histones, although the molecular weight and the charge of the proteins are significantly altered by their modification. The simultaneous elution of unmodified and labelled modified histones was detected by two types of gel electrophoresis and by autoradiography. In addition, the HPLC fractions did not display overlapping ladders of the multiply modified histones, as is commonly seen in one-dimensional electrophoretic analyses of unfractionated material. Hence individual bands could be unambiguously assigned. After in vitro labelling of isolated rat liver nuclei, the following ADP-ribosylated and unmodified histones were identified by HPLC and gel electrophoresis: histone H1(0), four histone H1 subfractions, histone H2A.1, histone H2A.2, oxidized histone H2A.2, histone H2A.X, histone H2A.Z, histone H2B, three histone H3 variants and histone H4.     

Comprehensive Profiling for Histone H4of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chro‐ matin, the regulation of gene activities and the diagnosis of diseases, and conducting in‐depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ‐Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂‐terminal tails were also identified after the LC‐MS/MS analysis of trypsin, Arg‐C, Glu‐C and chymotrypsin digests.