Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis

Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom‐up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom‐up MS on histones.     

Middle‐Down MS is Ready to Answer Complex Questions in Chromatin Biology

Histones are the most abundant protein family in the cells of complex organisms such as mammals and, together with DNA, they define the backbone of chromatin. Histone PTMs are key players of chromatin biology, as they function as anchors for proteins that bind and modulate chromatin readout, including gene expression. Middle‐down mass spectrometry (MS) has been optimized for about 10 years to study histone N‐terminal tails, but it has been rarely applied to identify the role of coexisting histone marks in biology. In this work, Jiang et al. used middle‐down MS to study the dynamics of coexisting PTMs on histone H4 in two breast cancer cell lines. 1 They found that overall serine 1 phosphorylation (S1ph) is mildly regulated during the cell cycle, but S1ph coexistence frequency with acetylations and methylations on the lysine residues of the N‐terminal tail is remarkably tuned during S phase and G2/M phase. Together, the team placed another benchmark proving that MS analysis of combinatorial histone PTMs provides a more comprehensive view on chromatin state than studying individual marks. We should then constantly question ourselves regarding the limitations of analyzing single PTMs when we attempt to define their effect on protein functions.     

Properly reading the histone code by MS‐based proteomics

Histone proteins are essential elements for DNA packaging. Their PTMs contribute in modeling chromatin structure and recruiting enzymes involved in gene regulation, DNA repair, and chromosome condensation. This fundamental aspect, together with the fact that histone PTMs can be epigenetically inherited through cell generations, enlightens their importance in chromatin biology, and the consequent necessity of having biochemical techniques for their characterization. Nanoflow LC coupled to MS (nanoLC‐MS) is the strategy of choice for protein PTM accurate quantification. However, histones require adjustments to the digestion protocol such as lysine derivatization to obtain suitable peptides for the analysis. nanoLC‐MS has numerous advantages, spanning from high confidence identification to possibility of high throughput analyses, but the peculiarity of the histone preparation protocol requires continuous monitoring with the most modern available technologies to question its reliability. The work of Meert et al. (Proteomics 2015, 15, 2966–2971) establishes which protocols lead to either incomplete derivatization or derivatization of undesired amino acid residues using a combination of high resolution MS and bioinformatics tools for the alignment and the characterization of nanoLC‐MS runs. As well, they identify a number of side reactions that could be potentially misinterpreted as biological PTMs.     

Quantitative assessment of chemical artefacts produced by propionylation of histones prior to mass spectrometry analysis

Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot‐gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain “Arg‐C like” digestion products that are more suitable for LC‐MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. 1 is a quantitative assessment of methyl‐esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059–2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl‐esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl‐esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation.     

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

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.     

Enhanced top-down characterization of histone post-translational modifications

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.     

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides.Chromatin is a highly organized and dynamic entity in cell nuclei, mostly composed of DNA and histone proteins. Its structure directly influences gene expression, DNA repair, and cell duplication events such as mitosis and meiosis (1). Histones are assembled in octamers named nucleosomes, wrapped by DNA every ∼200 base pairs. Histones are heavily modified by dynamic post-translational modifications (PTMs)¹, which affect chromatin structure because of their chemical properties and their ability to recruit chromatin modifier enzymes and binding proteins (2). Moreover, histone PTMs can be inherited through cell division and thus are crucial components of epigenetic memory (3). The function of histone PTMs has been extensively studied in the last 15–20 years, and several links have been found between aberrations of histone PTM levels and development of diseases (4, 5). Such discoveries revealed the importance of histone PTMs in fine-tuning cell phenotype. Because of this, technology has been rapidly evolving to investigate histone PTM relative abundance with higher accuracy and throughput.Mass spectrometry (MS)-based strategies have continuously evolved toward higher throughput and flexibility, allowing not only identification and quantification of single histone PTMs, but also their combinatorial patterns and even characterization of the intact proteins (reviewed in (6, 7)). For histone analysis, a widely adopted workflow for nano-liquid chromatography–tandem mass spectrometry (nLC-MS/MS) includes derivatization of lysine residue side chains with propionic anhydride, proteolytic digestion with trypsin, and subsequent derivatization of peptide N termini (8, 9). Such protocol leads to generation of ArgC-like peptides (only cleaved after arginine residues) after digestion. Moreover, propionylation of N termini increases peptide hydrophobicity, thereby improving LC retention of shorter ones, and thus the MS signal. Because of the high mass accuracy, sensitivity, and the possibility to perform label-free quantification MS has become the technique of choice, outperforming antibody-based strategies, to study both known and novel global histone PTMs.Several acquisition methods have been developed for MS analysis to accomplish different needs of identification and quantification (10). The most widely adopted in shotgun or discovery proteomics is the data-dependent acquisition (DDA) mode. Such acquisition method does not require any previous knowledge about the analyte, as it automatically selects precursor ions detectable at the full scan level in a given order (commonly from the most intense) to perform MS/MS fragmentation (11). Label-free quantification is performed at the full MS scan level by integrating the area of the LC peak from an extracted ion chromatogram of the precursor mass corresponding to the given peptide. On the other hand, the selected reaction monitoring (SRM) mode is the most widely used acquisition method in targeted proteomics. Such method performs cyclic precursor ion selection, MS/MS fragmentation, and product ion selection of a list of masses input by the user. Even though the method preparation is intuitively more complex than DDA, SRM is highly popular because of the high selectivity and sensitivity, which leads to more accurate label-free quantification (12). However, both methods have inevitable drawbacks; a DDA approach cannot perform accurate quantification of isobaric and co-eluting peptides, for example, KacQLATKAAR and KQLATKacAAR (histone H3 aa 9–17), as the fragment ions should be monitored through the entire peptide peak elution to define the ratio between the two similar analytes. On the contrary, an SRM experiment prevents future data mining of unpredicted peptides, and thus such method cannot be used for any classical PTM discovery. Therefore, LC-MS/MS analysis of histone peptides is commonly performed by integrating shotgun and targeted acquisition within the same MS method (13). This method requires previous knowledge about retention time and mass of co-eluting isobaric species, and tedious manual peak integration or dedicated software to deconvolute such complex raw data. Although this mixed MS mode is a powerful approach, the targeted sequences in the method reduce the duty cycle and number of DDA MS/MS spectra that can be acquired, making it far from ideal.Data independent acquisition (DIA) modes are a third option that recently gained popularity in proteomics (14, 15). Sequential window acquisition of all theoretical mass spectra (SWATH™)-MS is a data independent workflow that uses a first quadrupole isolation window to step across a mass range, collecting high resolution full scan composite MS/MS at each step and generating an ion map of fragments from all detectable precursor masses (15, 16). From such data set, a virtual SRM, or pseudo-SRM, can be performed by extracting the product ion chromatogram of a given peptide (17) with bioinformatics tools such as Peakview®, Skyline (18), or OpenSWATH™ (19). In order to define which fragment masses should be used to quantify a given peptide, a spectral library of identified peptides can be manually programmed, downloaded (if available), or generated by previous DDA experiments. In terms of quantification power, SWATH™ combines the advantages of both DDA and SRM, as it allows for MS/MS-based label-free quantification, discrimination of isobaric peptides, and subsequent data mining of unpredicted species.Histone proteins are an excellent target sample to test SWATH™, as the peptides are heavily modified by PTMs and often have isobaric proteoforms present. We analyzed with both DDA and SWATH™ two model systems: (1) extracted histones from untreated (pluripotent) and retinoic acid (RA) treated (differentiated) human embryonic stem cells (hESCs, strain H9), and (2) extracted histones from undifferentiated and differentiated mouse trophoblast stem cells (mTSCs). The results from the DDA experiment were used to evaluate the reproducibility of peptide retention time and the variety of species identified. For the SWATH™ analysis we focused on histone H3, as it is the histone with the highest variety of modified peptides (6). Results highlighted that such acquisition method provides sensitive and precise MS/MS-based quantification of both isobaric and nonisobaric peptides. Our data demonstrate that quantification at the MS/MS level is highly reproducible, and identification of the peptide elution profile is assisted by the high mass accuracy and the large number of overlapping elution profiles of the fragment ions. Moreover, we show that by using different fragment ions for MS/MS quantification we achieved similar quantification results. Thus, we used all unique fragment ions for a given species to provide a robust quantification method, where by unique is intended fragment ions that belong to only one of the possible isobaric peptide proteoforms. Taken together, we prove that SWATH™-MS is a reliable and simple-to-use acquisition method to perform epigenetic histone PTM analysis.     

Identification and Characterization of Propionylation at Histone H3 Lysine 23 in Mammalian Cells

Propionylation has been identified recently as a new type of protein post-translational modification. Bacterial propionyl-CoA synthetase and human histone H4 are propionylated at specific lysine residues that have been known previously to be acetylated. However, other proteins subject to this modification remain to be identified, and the modifying enzymes involved need to be characterized. In this work, we report the discovery of histone H3 propionylation in mammalian cells. Propionylation at H3 lysine Lys²³ was detected in the leukemia cell line U937 by mass spectrometry and Western analysis using a specific antibody. In this cell line, the propionylated form of Lys²³ accounted for 7%, a level at least 6-fold higher than in other leukemia cell lines (HL-60 and THP-1) or non-leukemia cell lines (HeLa and IMR-90). The propionylation level in U937 cells decreased remarkably during monocytic differentiation, indicating that this modification is dynamically regulated. Moreover, in vitro assays demonstrated that histone acetyltransferase p300 can catalyze H3 Lys²³ propionylation, whereas histone deacetylase Sir2 can remove this modification in the presence of NAD⁺. These results suggest that histone propionylation might be generated by the same set of enzymes as for histone acetylation and that selection of donor molecules (propionyl-CoA versus acetyl-CoA) may determine the difference of modifications. Because like acetyl-CoA, propionyl-CoA is an important intermediate in biosynthesis and energy production, histone H3 Lys²³ propionylation may provide a novel epigenetic regulatory mark for cell metabolism.     

Middle-down proteomics: a still unexploited resource for chromatin biology

Introduction: Analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has become a fundamental tool for the characterization of chromatin composition and dynamics. Histone PTMs benchmark several biological states of chromatin, including regions of active enhancers, active/repressed gene promoters and damaged DNA. These complex regulatory mechanisms are often defined by combinatorial histone PTMs; for instance, active enhancers are commonly occupied by both marks H3K4me1 and H3K27ac. The traditional bottom-up MS strategy identifies and quantifies short (aa 4–20) tryptic peptides, and it is thus not suitable for the characterization of combinatorial PTMs.

Areas covered: Here, we review the advancement of the middle-down MS strategy applied to histones, which consists in the analysis of intact histone N-terminal tails (aa 50–60). Middle-down MS has reached sufficient robustness and reliability, and it is far less technically challenging than PTM quantification on intact histones (top-down). However, the very few chromatin biology studies applying middle-down MS resulting from PubMed searches indicate that it is still very scarcely exploited, potentially due to the apparent high complexity of method and analysis.

Expert commentary: We will discuss the state-of-the-art workflow and examples of existing studies, aiming to highlight its potential and feasibility for studies of cell biologists interested in chromatin and epigenetics.     

Liquid chromatography mass spectrometry profiling of histones

Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RPLC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS.     

A new method for C-terminal sequence analysis in the proteomic era

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the amino or carboxy terminus are by far the most common types of PTMs in proteins. In contrast to the analysis of phosphorylation and glycosylation sites, relatively little attention has been paid to the development of approaches for the systematic analysis of proteolytic processing events. Here we present a new mass spectrometry (MS)-based strategy that allows the identification of the C-terminal sequence of proteins. The method can be directly applied to proteins cleaved with cyanogen bromide (CNBr) and purified either by SDS-PAGE, by two-dimensional (2D) PAGE or in solution, and it therefore eliminates the need for specific isolation of the C-terminal peptide. Using Shewanella oneidensis as a model system, we have demonstrated that this approach can be used for C-terminal sequence analysis at a proteomic scale. We also applied the method to study the C-terminal proteolytic processing of procardosin A.     

Interpreting the language of histone and DNA modifications

A major mechanism regulating the accessibility and function of eukaryotic genomes are the covalent modifications to DNA and histone proteins that dependably package our genetic information inside the nucleus of every cell. Formally postulated over a decade ago, it is becoming increasingly clear that post-translational modifications (PTMs) on histones act singly and in combination to form a language or ‘code’ that is read by specialized proteins to facilitate downstream functions in chromatin. Underappreciated at the time was the level of complexity harbored both within histone PTMs and their combinations, as well as within the proteins that read and interpret the language. In addition to histone PTMs, newly-identified DNA modifications that can recruit specific effector proteins have raised further awareness that histone PTMs operate within a broader language of epigenetic modifications to orchestrate the dynamic functions associated with chromatin. Here, we highlight key recent advances in our understanding of the epigenetic language encompassing histone and DNA modifications and foreshadow challenges that lie ahead as we continue our quest to decipher the fundamental mechanisms of chromatin regulation. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

Sulfo‐NHS‐SS‐biotin derivatization: A versatile tool for MALDI mass analysis of PTMs in lysine‐rich proteins

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo‐NHS‐SS‐biotin derivatization of lysine side chain can help to detect PTMs in lysine‐rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5‐lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.     

组蛋白泛素化修饰及其在DNA损伤应答中的作用

泛素化修饰是真核生物细胞内重要的翻译后修饰类型,通过调节蛋白质活性、稳定性和亚细胞定位广泛参与细胞内各项信号传导与代谢过程,对维持正常生命活动具有重要意义。组蛋白作为染色质中主要的蛋白成分,与DNA复制转录、修复等行为密切相关,是研究翻译后修饰的热点。DNA损伤后,组蛋白泛素化修饰通过调节核小体结构、激活细胞周期检查点、影响修复因子的招募与装配等诸多途径参与损伤应答。同时,组蛋白泛素化修饰还能调节其他位点翻译后修饰,并通过这种串扰(crosstalk)作用调节DNA损伤应答。本文介绍了组蛋白泛素化修饰的主要位点和相关组分(包括E3连接酶、去泛素化酶与效应分子),以及这些修饰作用共同编译形成的信号网络在DNA损伤应答中的作用,最后总结了目前该领域研究所面临的一些问题,以期为科研人员进一步探索组蛋白密码在DNA损伤应答中的作用提供参考。     

A tool to evaluate correspondence between extraction ion chromatographic peaks and peptide‐spectrum matches in shotgun proteomics experiments

Chromatographed peptide signals form the basis of further data processing that eventually results in functional information derived from data‐dependent bottom‐up proteomics assays. We seek to rank LC/MS parent ions by the quality of their extracted ion chromatograms. Ranked extracted ion chromatograms act as an intuitive physical/chemical preselection filter to improve the quality of MS/MS fragment scans submitted for database search. We identify more than 4900 proteins when considering detector shifts of less than 7 ppm. High quality parent ions for which the database search yields no hits become candidates for subsequent unrestricted analysis for PTMs. Following this rational approach, we prioritize identification of more than 5000 spectrum matches from modified peptides and confirmed the presence of acetylaldehyde‐modified His/Lys. We present a logical workflow that scores data‐dependent selected ion chromatograms and leverage information about semianalytical LC/LC dimension prior to MS. Our method can be successfully used to identify unexpected modifications in peptides with excellent chromatography characteristics, independent of fragmentation pattern and activation methods. We illustrate analysis of ion chromatograms detected in two different modes by RF linear ion trap and electrostatic field orbitrap.