Enrichment of histones from patient samples for mass spectrometry-based analysis of post-translational modifications.

Aberrations in histone post-translational modifications (PTMs) have been implicated with the development of numerous pathologies, including cancer. Therefore, profiling histone PTMs in patient samples could provide information useful for the identification of epigenetic biomarkers, as well as for the discovery of potential novel targets. While antibody-based methods have been traditionally employed to analyze histone PTM in clinical samples, mass spectrometry (MS) can provide a more comprehensive, unbiased and quantitative view on histones and their PTMs. To combine the power of MS-based methods and the potential offered by histone PTM profiling of clinical samples, we have recently developed a series of methods for the extraction and enrichment of histones from different types of patient samples, including formalin-fixed paraffin-embedded tissues, fresh- and optimal cutting temperature-frozen tissues, and primary cells. Here, we provide a detailed description of these protocols, together with indications on the expected results and the most suitable workflow to be used downstream of each procedure.

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.

Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples.

PURPOSE: Profiling histone posttranslational modifications (PTMs) in clinical samples holds great potential for the identification of epigenetic biomarkers and the discovery of novel epigenetic targets. MS-based approaches to analyze histone PTMs in clinical samples usually rely on SDS-PAGE separation following histone enrichment in order to eliminate detergents and further isolate histones. However, this limits the digestions options and hence the modification coverage. EXPERIMENTAL DESIGN AND RESULTS: The aim of this study is the implementation of a procedure involving acetone protein precipitation followed by histone enrichment through a C18 StageTip column to obtain histone preparations suitable for various in-solution digestion protocols. Among them, the Arg-C digestion, which allows profiling histone H4 modifications, and the Prop-PIC method, which improves the detection of short and hydrophilic peptides, are tested. This approach is validated on different types of samples, including formalin-fixed paraffin-embedded pathology tissues, and employed to profile histone H4 modifications in cancer samples and normal tissues, identifying previously reported differences, as well as novel ones. CONCLUSIONS AND CLINICAL RELEVANCE: This protocol widens the number of applications available in the toolbox of clinical epigenomics, allowing the investigation of a larger spectrum of histone marks in patient samples.

Profiling of Epigenetic Features in Clinical Samples Reveals Novel Widespread Changes in Cancer.

Aberrations in histone post-translational modifications (PTMs), as well as in the histone modifying enzymes (HMEs) that catalyze their deposition and removal, have been reported in many tumors and many epigenetic inhibitors are currently under investigation for cancer treatment. Therefore, profiling epigenetic features in cancer could have important implications for the discovery of both biomarkers for patient stratification and novel epigenetic targets. In this study, we employed mass spectrometry-based approaches to comprehensively profile histone H3 PTMs in a panel of normal and tumoral tissues for different cancer types, identifying various changes, some of which appear to be a consequence of the increased proliferation rate of tumors, while others are cell-cycle independent. Histone PTM changes found in tumors partially correlate with alterations of the gene expression profiles of HMEs obtained from publicly available data and are generally lost in culture conditions. Through this analysis, we identified tumor- and subtype-specific histone PTM changes, but also widespread changes in the levels of histone H3 K9me3 and K14ac marks. In particular, H3K14ac showed a cell-cycle independent decrease in all the seven tumor/tumor subtype models tested and could represent a novel epigenetic hallmark of cancer. .