Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 -20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

Fungal peroxygenases are novel extracellular heme-thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H2O2 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 H2O2-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 H218O2 resulted in complete incorporation of 18O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n-hexane-1,1,1,2,2,3,3-D7 showed a large intramolecular deuterium isotope effect [(kH/kD)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 × 1011 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 (> C16) 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.

BackgroundDuring the process of spermatogenesis, male germ cells undergo dramatic chromatin reorganization, whereby most histones are replaced by protamines, as part of the pathway to compact the genome into the small nuclear volume of the sperm head. Remarkably, approximately 90 % (human) to 95 % (mouse) of histones are evicted during the process. An intriguing hypothesis is that post-translational modifications (PTMs) decorating histones play a critical role in epigenetic regulation of spermatogenesis and embryonic development following fertilization. Although a number of specific histone PTMs have been individually studied during spermatogenesis and in mature mouse and human sperm, to date, there is a paucity of comprehensive identification of histone PTMs and their dynamics during this process.ResultsHere we report systematic investigation of sperm histone PTMs and their dynamics during spermatogenesis. We utilized “bottom-up” nanoliquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) to identify histone PTMs and to determine their relative abundance in distinct stages of mouse spermatogenesis (meiotic, round spermatids, elongating/condensing spermatids, and mature sperm) and in human sperm. We detected peptides and histone PTMs from all four canonical histones (H2A, H2B, H3, and H4), the linker histone H1, and multiple histone isoforms of H1, H2A, H2B, and H3 in cells from all stages of mouse spermatogenesis and in mouse sperm. We found strong conservation of histone PTMs for histone H3 and H4 between mouse and human sperm; however, little conservation was observed between H1, H2A, and H2B. Importantly, across eight individual normozoospermic human semen samples, little variation was observed in the relative abundance of nearly all histone PTMs.ConclusionIn summary, we report the first comprehensive and unbiased analysis of histone PTMs at multiple time points during mouse spermatogenesis and in mature mouse and human sperm. Furthermore, our results suggest a largely uniform histone PTM signature in sperm from individual humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0072-6) contains supplementary material, which is available to authorized users.

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) ×107 M−1s−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 five orders of magnitude and correlated linearly with bond dissociation enthalpies of strong C–H bond substrates with a log k2’ vs. BDE slope of ~ 0.4. However, the hydroxylation rate was insensitive to C–H BDE below 90 kcal/mol, similar to the behavior of the t-butoxy 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 FeIVO–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.

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.

Recent advances in high resolution mass spectrometry (MS) instrumentation and semi-automated software have led to a push toward the use of MS-based methods for quality control (QC) testing of therapeutic proteins in a cGMP environment. The approach that is most commonly being proposed for this purpose is known as the multi-attribute method (MAM). MAM is a promising approach that provides some distinct benefits compared to conventional methods currently used for QC testing of protein therapeutics, such as CEX, HILIC, and CE-SDS. Because MS-based methods have not been regularly used in this context in the past, new scientific and regulatory questions should be addressed prior to the final stages of implementation. We have categorized these questions into four major aspects for MAM implementation in a cGMP environment for both new and existing products: risk assessment, method validation, capabilities and specificities of the New Peak Detection (NPD) feature, and comparisons to conventional methods. This perspective outlines considerations for each of these main points and suggests approaches to help address potential issues.

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 labelfree 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 spectrometrybased quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples. Molecular & Cellular

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 13 C-labeled UDP-GlcNAc from 13 C 6 -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 OGlcNAcylation 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. Molecular &