Comprehensive histone epigenetics : a mass spectrometry based screening assay to measure epigenetic toxicity

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 role of small proteins in Burkholderia cenocepacia J2315 biofilm formation, persistence and intracellular growth

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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Removing the hidden data dependency of DIA with predicted spectral libraries

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. Here, it is shown 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

An experimental design to extract more information from MS-based histone studies

Histone-based chromatin organization paved the way for eukaryotic genome complexity. Because of their key role in information management, the histone posttranslational modifications (hPTM), which mediate their function, have evolved into an alphabet that has more letters than there are amino acids, together making up the ‘‘histone code’’. The resulting combinatorial complexity is manifold higher than what is usually encountered in proteomics. Consequently, a considerably bigger part of the acquired MSMS spectra remains unannotated to date. Adapted search parameters can dig deeper into the dark histone ion space, but the lack of false discovery rate (FDR) control and the high level of ambiguity when searching combinatorial PTMs makes it very hard to assess whether the newly assigned ions are informative. Therefore, we propose an easily adoptable time-lapse enzymatic deacetylation (HDAC1) of a commercial histone extract as a quantify-first strategy that allows isolating ion populations of interest, when studying e.g. acetylation on histones, that currently remain in the dark. By adapting search parameters to study potential issues in sample preparation, data acquisition and data analysis, we stepwise managed to double the portion of annotated precursors of interest from 10.5% to 21.6%. This strategy is intended to make up for the lack of validated FDR control and has led to several adaptations of our current workflow that will reduce the portion of the dark histone ion space in the future. Finally, this strategy can be applied with any enzyme targeting a modification of interes

Histone sample preparation for bottom-up mass spectrometry : a roadmap to informed decisions

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(R), Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423

Analyzing data independent acquisition mass spectrometry with noiseless ion-networks

Data-independent acquisition (DIA) mass spectrometry (MS) has introduced deter-ministic, periodic and simultaneous acquisition of all fragment ions. Despite the chimeric side-effects associated with this unprecedented data integrity, DIA data analysis approaches still use conventional spectra and extracted ion chromatograms (XICs) that represent individual precursors and fragments. Here, we introduce ion-networks, an alternative data format wherein nodes correspond to reproducible fragment ions from multiple runs and edges correspond to consistent co-elution. Each ion-network represents a complete experiment and computationally eliminates chimericy based on reproducibility without sacrificing data integrity

hSWATH : unlocking SWATH’s full potential for an untargeted histone perspective

Mass spectrometry (MS) has become the technique of choice for large-scale analysis of histone post-translational modifications (hPTMs) and their combinatorial patterns, especially in untargeted settings where novel discovery-driven hypotheses are being generated. However, MS-based histone analysis requires a distinct sample preparation, acquisition, and data analysis workflow when compared to traditional MS-based approaches. To this end, sequential window acquisition of all theoretical fragment ion spectra (SWATH) has great potential, as it allows for untargeted accurate identification and quantification of hPTMs. Here, we present a complete SWATH workflow specifically adapted for the untargeted study of histones (hSWATH). We assess its validity on a technical dataset of time-lapse deacetylation of a commercial histone extract using HDAC1, which contains a ground truth, i.e., acetylated substrate peptides reduce in intensity. We successfully apply this workflow in a biological setting and subsequently investigate the differential response to HDAC inhibition in different breast cancer cell lines