Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

University of Pennsylvania

Fungal peroxygenases are novel extracellular heme-thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H(2)O(2) as a cosubstrate. Little is known about the physiological role or the catalytic mechanism of these enzymes. We have found that the peroxygenase secreted by Agrocybe aegerita catalyzes the H(2)O(2)-dependent hydroxylation of linear alkanes at the 2-position and 3-position with high efficiency, as well as the regioselective monooxygenation of branched and cyclic alkanes. Experiments with n-heptane and n-octane showed that the hydroxylation proceeded with complete stereoselectivity for the (R)-enantiomer of the corresponding 3-alcohol. Investigations with a number of model substrates provided information about the route of alkane hydroxylation: (a) the hydroxylation of cyclohexane mediated by H(2)(18)(2) resulted in complete incorporation of (18)O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n-hexane-1,1,1,2,2,3,3-D(7) showed a large intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 16.0 ± 1.0 for 2-hexanol and 8.9 ± 0.9 for 3-hexanol; and (c) the hydroxylation of the radical clock norcarane led to an estimated radical lifetime of 9.4 ps and an oxygen rebound rate of 1.06 × 10(11) s(-1). These results point to a hydrogen abstraction and oxygen rebound mechanism for alkane hydroxylation. The peroxygenase appeared to lack activity on long-chain alkanes (> C(16)) and highly branched alkanes (e.g. tetramethylpentane), but otherwise exhibited a broad substrate range. It may accordingly have a role in the bioconversion of natural and anthropogenic alkane-containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood.

Selective hydroxylation of alkanes by an extracellular fungal peroxygenase.

The extracellular heme-thiolate peroxygenase from Agrocybe aegerita (AaeAPO) has been shown to hydroxylate alkanes and numerous other substrates using hydrogen peroxide as the terminal oxidant. We describe the kinetics of formation and decomposition of AaeAPO compound I upon its reaction with mCPBA. The UV-vis spectral features of AaeAPO-I (361, 694 nm) are similar to those of chloroperoxidase-I and the recently described cytochrome P450-I. The second-order rate constant for AaeAPO-I formation was 1.0 (±0.4) × 10(7) M(-1) s(-1) at pH 5.0, 4 °C. The relatively slow decomposition rate, 1.4 (±0.03) s(-1), allowed the measurement of its reactivity toward a panel of substrates. The observed rate constants, k2', spanned 5 orders of magnitude and correlated linearly with bond dissociation enthalpies (BDEs) of strong C-H bond substrates with a log k2' vs BDE slope of ∼0.4. However, the hydroxylation rate was insensitive to a C-H BDE below 90 kcal/mol, similar to the behavior of the tert-butoxyl radical. The shape and slope of the Brønsted-Evans-Polanyi plot indicate a symmetrical transition state for the stronger C-H bonds and suggest entropy control of the rate in an early transition state for weaker C-H bonds. The AaeAPO-II Fe(IV)O-H BDE was estimated to be ∼103 kcal/mol. All results support the formation of a highly reactive AaeAPO oxoiron(IV) porphyrin radical cation intermediate that is the active oxygen species in these hydroxylation reactions.

Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I.

Driving force for oxygen-atom transfer by heme-thiolate enzymes.

MS-based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in RP LC, and such procedure also hampers the discovery of natural propionylation events. In this work we tested 12 commercially available anhydrides, selected based on their safety and hydrophobicity. Performance was evaluated in terms of yield of the reaction, MS/MS fragmentation efficiency, and drift in retention time using the following samples: (i) a synthetic unmodified histone H3 tail, (ii) synthetic modified histone peptides, and (iii) a histone extract from cell lysate. Results highlighted that seven of the selected anhydrides increased peptide retention time as compared to propionic, and several anhydrides such as benzoic and valeric led to high MS/MS spectra quality. However, propionic anhydride derivatization still resulted, in our opinion, as the best protocol to achieve high MS sensitivity and even ionization efficiency among the analyzed peptides.

Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom-up proteomics PTM analysis.

During spermiogenesis, the postmeiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyperacetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyperacetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. Chromatin immunoprecipitation followed by sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence of a prominent transcriptional role for BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed and chromatin is compacted during spermiogenesis.

Characterization of BRD4 during mammalian postmeiotic sperm development.

A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapid-mixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pKa of 10.0 for the Fe(IV)-O-H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C-H and phenolic substrates, with second-order rate constants for C-H and O-H bond scission in the range of 10-10(7) M(-1)⋅s(-1). Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C-H and O-H bond scission.

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive.

Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.

EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra.

Metal-free heteroatom-doped carbocatalysts with a high surface area are desirable for catalytic reactions. In this study, we found an efficient strategy to prepare nitrogen, phosphorus, and sulfur co-doped hollow carbon shells (denote as NPS-HCS) with a surface area of 1020 m(2)  g(-1) . Using a poly(cyclotriphosphazene-co-4,4'-sulfonyldiphenol) (PZS) shell as carbon source and N, P, S-doping source, and the ZIF-67 core as structural template as well as extra N-doping source, NPS-HCS were obtained with a high surface area and superhydrophilicity. All these features render the prepared NPS-HCS a superior metal-free carbocatalyst for the selective oxidation of aromatic alkanes in aqueous solution. This study provides a reliable and facile route to prepare doped carbocatalysts with enhanced catalytic properties.

Nitrogen, Phosphorus, and Sulfur Co-Doped Hollow Carbon Shell as Superior Metal-Free Catalyst for Selective Oxidation of Aromatic Alkanes.

Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13C-labeled UDP-GlcNAc from 13C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time.

A novel quantitative mass spectrometry platform for determining protein O-GlcNAcylation dynamics.

We report an efficient and controllable route to prepare magnetic tubular nanoreactors composed of mesoporous SiO2 shell and iron-noble metal nanoparticles residing inside. The key of this method is to design and fabricate Fe nanoparticles encapsulated in mesoporous SiO2 shell. Making use of galvanic replacement reaction between Fe and noble metal ions, all the noble metal nanoparticles are loaded inside the mesoporous SiO2 shell, and thus nanoreactors are formed. Taking Pd as an example, the prepared Pd-Fe@meso-SiO2 tubular nanoreactor exhibits highly catalytic activity and excellent reusability for styrene hydrogenation under mild conditions. This study provides a facile route to fabricate magnetic nanoreactors with enhanced catalytic properties.

Xiaoshi Wang

Epigenetics Program,
Department of Biochemistry and Biophysics,
Perelman School of Medicine,
Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine

University of Pennsylvania

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Xiaoshi Wang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Epigenetics Program,Department of Biochemistry and Biophysics,Perelman School of Medicine,Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine

University of Pennsylvania

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Epigenetics Program,
Department of Biochemistry and Biophysics,
Perelman School of Medicine,
Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine