Abstract Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment-between-runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols, while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX ® , Genovis), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data is available via ProteomeXchange with identifier PXD024423.
Burkholderia cenocepacia infections are difficult to treat due to resistance, biofilm formation and persistence. B. cenocepacia strain J2315 has a large multi-replicon genome (8.06 Mb) and the function of a large fraction of (conserved) hypothetical genes remains elusive. The goal of the present study is to elucidate the role of small proteins in B. cenocepacia, focusing on genes smaller than 300 base pairs of which the function is unknown. Almost 10% (572) of the B. cenocepacia J2315 genes are smaller than 300 base pairs and more than half of these are annotated as coding for hypothetical proteins. For 234 of them no similarity could be found with non-hypothetical genes in other bacteria using BLAST. Using available RNA sequencing data obtained from biofilms, a list of 27 highly expressed B. cenocepacia J2315 genes coding for small proteins was compiled. For nine of them expression in biofilms was also confirmed using LC-MS based proteomics and/or expression was confirmed using eGFP translational fusions. Overexpression of two of these genes negatively impacted growth, whereas for four others overexpression led to an increase in biofilm biomass. Overexpression did not have an influence on the MIC for tobramycin, ciprofloxacin or meropenem but for five small protein encoding genes, overexpression had an effect on the number of persister cells in biofilms. While there were no significant differences in adherence to and invasion of A549 epithelial cells between the overexpression mutants and the WT, significant differences were observed in intracellular growth/survival. Finally, the small protein BCAM0271 was identified as an antitoxin belonging to a toxin-antitoxin module. The toxin was found to encode a tRNA acetylase that inhibits translation. In conclusion, our results confirm that small proteins are present in the genome of B. cenocepacia J2315 and indicate that they are involved in various biological processes, including biofilm formation, persistence and intracellular growth.
LC-MS/MS extends on the MALDI-TOF ZooMS approach by providing fragmentation spectra for each peptide. However, ancient bone samples generate sparse datasets containing only a few collagen proteins, rendering target-decoy strategies unusable and increasing uncertainty in peptide annotation. ClassiCOL embraces and even extends this ambiguity using a novel isoBLAST approach. The exhaustive set of potential peptide candidates created in this way is then used to retain or reject different potential paths at each taxonomic branching point down to the taxonomic level attainable with the sample information, always allowing for potential mixtures in the process. As an end point, all considered ambiguity is graphically represented with a clear prioritization of the species in the sample. Using public as well as in-house data, we demonstrate the performance of this universal postprocessing approach on different instruments and explore the possibility of identifying genetic as well as sample mixtures. Diet reconstruction from 40,000 year old cave hyena coprolites illustrates the exciting potential of this approach.
Chikungunya virus (CHIKV) is an arbovirus with a global spread and significant public health impact. It is a positive stranded RNA alphavirus belonging to the Togaviridae family. However, many questions about the replication cycle of CHIKV remain unanswered. The entry process of CHIKV is not completely understood nor are the associated virus-receptor interactions fully identified. Here, we designed an affinity purification mass spectrometry coupled approach that allowed the identification of factors that facilitate entry of CHIKV in human cells. The identified entry factors were further validated using CRISPR/Cas9. In HEK293T cells we identified the CD147 protein complex as an entry factor for CHIKV. We further showed the involvement of the CD147 protein complex in the replication cycle of related alphaviruses. Interestingly, CD147 contains similar protein domains as the previously identified alphavirus entry factor MXRA8.
Evidence of the involvement of epigenetics in pathologies such as cancer, diabetes, and neurodegeneration has increased global interest in epigenetic modifications. For nearly thirty years, it has been known that cancer cells exhibit abnormal DNA methylation patterns. In contrast, the large-scale analysis of histone post-translational modifications (hPTMs) has lagged behind because classically, histone modification analysis has relied on site specific antibody-based techniques. Mass spectrometry (MS) is a technique that holds the promise to picture the histone code comprehensively in a single experiment. Therefore, we developed an MS-based method that is capable of tracking all possible hPTMs in an untargeted approach. In this way, trends in single and combinatorial hPTMs can be reported and enable prediction of the epigenetic toxicity of compounds. Moreover, this method is based on the use of human cells to provide preliminary data, thereby omitting the need to sacrifice laboratory animals. Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort. Still, this novel toxicoepigenetic assay and the data it generates holds great potential for, among others, pharmaceutical industry, food science, clinical diagnostics and, environmental toxicity screening. There is a growing interest in epigenetic modifications, and more specifically in histone post-translational modifications (hPTMs). We describe an MS-based workflow that is capable of tracking all possible hPTMs in an untargeted approach that makes use of human cells. Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort.
The information that defines all organisms, including humans, is encoded in their DNA, which altogether comprises the genotype. Throughout life, from initial organ formation to later lifestyle exposures, different genes are epigenetically activated and transcribed into mRNA, which is in turn translated into proteins that make up the final phenotype. Therefore, proteins are the most prominent source of phenotypic information and have consequently been exhaustively studied in different contexts, including clinical biomarker discovery for diseases. Despite the fact that proteins are extremely stable biomolecules that can hold phenotypic information over thousands of years, their potential in forensic and archaeological research has not yet been fully exploited. At least in part, this is due to the fact that the field of proteomics has mainly focused on fundamental molecular biology and medical applications, developing tools to maximize the number of peptide identifications in tryptic digests of (liquid) biopsies and cell cultures, which are very different from the sparse and heavily modified peptide mixtures often originating from unknown sources that need to be identified in forensic and archaeological contexts. Here, we describe the growing interest and recent developments in forensic proteomics and illustrate how palaeoproteomics, its equivalent for more ancient times, provides valuable methodological insights for future forensic applications.
Data-Independent Acquisition (DIA) generates comprehensive yet complex mass spectrometric data, which imposes the use of data-dependent acquisition (DDA) libraries for deep peptide-centric detection. We here show that DIA can be redeemed from this dependency by combining predicted fragment intensities and retention times with narrow window DIA. This eliminates variation in library building and omits stochastic sampling, finally making the DIA workflow fully deterministic. Especially for clinical proteomics, this has the potential to facilitate inter-laboratory comparison. Significance of the Study Data-independent acquisition (DIA) is quickly developing into the most comprehensive strategy to analyse a sample on a mass spectrometer. Correspondingly, a wave of data analysis strategies has followed suit, improving the yield from DIA experiments with each iteration. As a result, a worldwide wave of investments in DIA is already taking place in anticipation of clinical applications. Yet, there is considerable confusion about the most useful and efficient way to handle DIA data, given the plethora of possible approaches with little regard for compatibility and complementarity. In our manuscript, we outline the currently available peptide-centric DIA data analysis strategies in a unified graphic called the DIAmond DIAgram. This leads us to an innovative and easily adoptable approach based on predicted spectral information. Most importantly, our contribution removes what is arguably the biggest bottleneck in the field: the current need for Data Dependent Acquisition (DDA) prior to DIA analysis. Fractionation, stochastic data acquisition, processing and identification all introduce bias in the library. By generating libraries through data independent, i.e. deterministic acquisition, stochastic sampling in the DIA workflow is now fully omitted. This is a crucial step towards increased standardization. Additionally, our results demonstrate that a proteome-wide predicted spectral library can surrogate an exhaustive DDA Pan-Human library that was built based on 331 prior DDA runs.
Abstract The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development.
So far, no 87 Sr/86 Sr mobility studies have been done for Neolithic remains from Belgium and information on the Sr isotopic variability in the region is scarce. This study aims to explore mobility in a Final Neolithic population from the funerary cave 'Grotte de La Faucille', contribute to the understanding of the isotopic composition of bioavailable Sr in Belgium, assess evidence for male mobility using proteomic analysis, and explore possible places of origin for nonlocal individuals.The 87 Sr/86 Sr isotope ratio of dental enamel from six adults and six juveniles was determined. Liquid chromatography mass spectrometry-based protein analysis was employed to identify individuals of male biological sex. 87 Sr/86 Sr of micromammal teeth, snail shells, and modern plants from three geological areas in Belgium were measured to establish isotopic signatures for bioavailable strontium. Nonlocality was assessed by comparing human 87 Sr/86 Sr isotope ratios to the 87 Sr/86 Sr range for bioavailable Sr.Four individuals yielded 87 Sr/86 Sr isotope ratios consistent with a nonlocal origin. No statistical differences were found between adults and juveniles. Three males were detected in the sample set, of which two show nonlocal 87 Sr/86 Sr values.This study provides evidence for mobility in Final Neolithic Belgium. The four nonlocal 87 Sr/86 Sr signatures correspond with the 87 Sr/86 Sr of bio-available Sr in Dutch South Limburg, the Black Forest in Southwest Germany, and regions of France, such as parts of the Paris Basin and the Vosges. The results support the ruling hypothesis of connections with Northern France, brought to light by archeological research.
Abstract Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. To prove the applicability and performance, we first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate that our workflow is capable of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose-response study was performed on a proof-of-concept library of ten compounds i) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), ii) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), iii) classified as non-embryotoxic by ECVAM (Penicillin G), and iv) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine). In conclusion, we show that toxicoepigenetic screening on histones is feasible and yields very rich data that holds potential, not only for applications in the pharmaceutical industry, but also for environmental toxicity and food safety.
