Analysis of human histone H4 by capillary electrophoresis in a pullulan-coated capillary,LC-ESI-MS and MALDI-TOF-MS

The object of the present study was the analysis of the human histone H4 (a core histone) in order to evaluate the state of its acetylation. Capillary electrophoresis (CE) using a pullulan-coated capillary provides a rapid and efficient approach to the separation of monoacetylated, diacetylated and triacetylated H4 isoforms from human cells. By using a simple running buffer of 100 mM triethanolamine-phosphate solution at pH 2.5 and exploiting the effectiveness of pullulan-based coverage in preventing adsorptive phenomena, the separation of the differently acetylated isoforms was achieved in less than 15 min with high efficiency and reproducibility. The proposed method was for the first time applied in the analysis of histone H4 fractions obtained from cell lines treated with different histone deacetylase (HDAC) inhibitors, used as potential anticancer drugs. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis demonstrated that the acetylation occurred in the histone H4 tail, whereas the CE separation allowed for a fast determination of the percentages of H4 acetylated isoforms in real samples; the results were in agreement with those obtained from liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) analysis. Therefore, the proposed CE method is a useful complementary support to the hyphenated techniques for the rapid monitoring of the activity of HDAC inhibitors.     

Defective histone H3K27 trimethylation modification in embryos derived from heated mouse sperm

The mouse sperm genome is resistant to in vitro heat treatment, and embryos derived from heated sperm can support full-term embryonic development, but the blastocyst rate and implantation rate are lower compared to embryos derived from fresh sperm. In the present study, the patterns of DNA methylation, histone H4K12 (ACH4K12) acetylation, H3K9 trimethylation (H3K9-TriM), and H3K27 trimethylation (H3K27-TriM) in preimplantation embryos derived from 65 °C-heated sperm were investigated. Although no evident changes in global DNA methylation, histone H4K12 (ACH4K12) acetylation, and H3K9 trimethylation (H3K9-TriM) were found, significantly lower levels of H3K27-TriM, which was thought to be one of the reasons for low efficiency of mouse cloning, were found in the inner cell mass of heated-sperm derived blastocysts. Thus, defective modification of H3K27-TriM might contribute to compromised development of embryos derived from heated sperm.     

组蛋白H3赖氨酸27和36位甲基化修饰的鉴定

采用液相色谱-质谱联用技术结合生物信息学分析手段,研究Hela细胞组蛋白H3赖氨酸(Lysine (K))K27和K36位点带有甲基化和二甲基化修饰的多肽鉴定,通过二级质谱碎片解析和二级碎片丰度分析,对组蛋白H3赖氨酸K27和K36上甲基化和二甲基化修饰进行了鉴定和分析。     

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.     

Structure and binding of the H4 histone tail and the effects of lysine 16 acetylation

The H4 histone tail plays a critical role in chromatin folding and regulation--it mediates strong interactions with the acidic patch of proximal nucleosomes and its acetylation at lysine 16 (K16) leads to partial unfolding of chromatin. The molecular mechanism associated with the H4 tail/acidic patch interactions and its modulation via K16 acetylation remains unknown. Here we employ a combination of molecular dynamics simulations, molecular docking calculations, and free energy computations to investigate the structure of the H4 tail in solution, the binding of the H4 tail with the acidic patch, and the effects of K16 acetylation. The H4 tail exhibits a disordered configuration except in the region Ala15-Lys20, where it exhibits a strong propensity for an α-helical structure. This α-helical region is found to dock very favorably into the acidic patch groove of a nucleosome with a binding free energy of approximately -7 kcal mol(-1). We have identified the specific interactions that stabilize this binding as well as the associated energetics. The acetylation of K16 is found to reduce the α-helix forming propensity of the H4 tail and K16's accessibility for mediating external interactions. More importantly, K16 acetylation destabilizes the binding of the H4 tail at the acidic patch by mitigating specific salt bridges and longer-ranged electrostatic interactions mediated by K16. Our study thus provides new microscopic insights into the compaction of chromatin and its regulation via posttranslational modifications of histone tails, which could be of interest to chromatin biology, cancer, epigenetics, and drug design.     

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。     

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.     

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.     

Mapping Post-translational Modifications of Histones H2A, H2B and H4 in Schizosaccharomyces pombe

Core histones are known to carry a variety of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation and ubiquitination, which play important roles in the epigenetic control of gene expression. The nature and biological functions of these PTMs in histones from plants, animals and budding yeast have been extensively investigated. In contrast, the corresponding studies for fission yeast were mainly focused on histone H3. In the present study, we applied LC-nano-ESI-MS/MS, coupled with multiple protease digestion, to identify PTMs in histones H2A, H2B and H4 from Schizosaccharomyces pombe (S. pombe), the typical model organism of fission yeast. Various protease digestions provided high sequence coverage for PTM mapping, and accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from tri-methylation. Many modification sites conserved in other organisms were identified in S. pombe. In addition, some unique modification sites, including N-terminal acetylation in H2A and H2B as well as K123 acetylation in H2A.β, were observed. Our results provide a comprehensive picture of the PTMs of histones H2A, H2B and H4 in S. pombe, which serves as a foundation for future investigations on the regulation and functions of histone modifications in this important model organism.     

Epsilon -N,N,N-trimethyllysine-specific ions in matrix-assisted laser desorption/ionization-tandem mass spectrometry

Epsilon -N,N,N-trimethyllysine (K(me3)) is a component of a number of proteins and plays an important role in the expression of their biological functions. Trimethylation, which causes an incremental increase in mass of 42.0470 Da from that of the corresponding MH(+) ion, cannot be distinguished from the acetylation (+42.0106 Da), which also occurs on epsilon-amino groups of Lys or alpha-amino groups in many proteins, without high-accuracy mass measurement which is accurate to within the second decimal place. MALDI-MS and MS/MS have been applied for the analyses of post-translational modifications of histone H3, which is known to contain both multiple acetylation and methylation sites in its sequence. During the measurements of the modified peptides, a novel fragmentation which involves the loss of trimethylamine from K(me3) was found. This characteristic fragmentation, which was observed to produce ions separated by 59 Da from the conventional precursor ion or sequence ions, would be useful for probing K(me3) units in the sequence.     

Reactivity and adduct formation of a polyaromatic hydrocarbon, 7-bromomethylbenz[a]anthracene, with chromatin histone proteins

The alkylation of histones by the direct-acting carcinogen 7-bromomethylbenz[a]anthracene was demonstrated both in vivo and in vitro. The relative molar reactivity for mouse liver histones in vivo was H3 greater than H1 greater than H2b greater than H4 greater than H2a. The in vitro modification of histone H3 was examined in detail. Amino acid adducts stable to acid hydrolysis were separated after acetylation by reversed-phase high-performance liquid chromatography and characterized using ultraviolet absorbance spectra and synthetic amino acid adduct standards. Three major adducts were observed and tentatively identified as cysteinyl, lysyl and histidinyl adducts of histone H3.     

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.