Progress in epigenetic histone modification analysis by mass spectrometry for clinical investigations (original) (raw)
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Expert review of proteomics, 2016
Histone post-translational modifications (hPTMs) contribute to the regulation of gene expression and increasing evidence links them to the development of various pathologies, highlighting their potential as biomarkers for prognostic, diagnostic and therapeutic applications. Mass spectrometry (MS) has emerged as a powerful analytical tool for hPTM analysis, which has also been applied to the analysis of epigenetic aberrations in diseases. However, the potential offered by the MS-based hPTM analysis of clinical samples for epigenetic biomarker discovery has been left largely unexploited. This article summarizes the contribution of MS-based approaches to clinical epigenetics, with a special focus on the PAThology tissue analysis of Histones by Mass Spectrometry (PAT-H-MS) approach--which represents the first application of MS-based hPTM analysis to formalin-fixed paraffin-embedded clinical samples--discussing its strengths and limitations, as well as possible implementations.
Genome Research, 2019
DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We d...
Molecular & cellular proteomics : MCP, 2015
Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from FFPE tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAT-H-...
The contribution of mass spectrometry-based proteomics to understanding epigenetics
Epigenomics, 2016
Chromatin is a macromolecular complex composed of DNA and histones that regulate gene expression and nuclear architecture. The concerted action of DNA methylation, histone post-translational modifications and chromatin-associated proteins control the epigenetic regulation of the genome, ultimately determining cell fate and the transcriptional outputs of differentiated cells. Deregulation of this complex machinery leads to disease states, and exploiting epigenetic drugs is becoming increasingly attractive for therapeutic intervention. Mass spectrometry (MS)-based proteomics emerged as a powerful tool complementary to genomic approaches for epigenetic research, allowing the unbiased and comprehensive analysis of histone post-translational modifications and the characterization of chromatin constituents and chromatin-associated proteins. Furthermore, MS holds great promise for epigenetic biomarker discovery and represents a useful tool for deconvolution of epigenetic drug targets. Here...
Data in brief, 2016
Aberrant histone post-translational modifications (hPTMs) have been implicated with various pathologies, including cancer, and may represent useful epigenetic biomarkers. The data described here provide a mass spectrometry-based quantitative analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE) tissues, from which histones were extracted through the recently developed PAT-H-MS method. First, we analyzed FFPE samples from mouse spleen and liver or human breast cancer up to six years old, together with their corresponding fresh frozen tissue. We then combined the PAT-H-MS approach with a histone-focused version of the super-SILAC strategy-using a mix of histones from four breast cancer cell lines as a spike-in standard- to accurately quantify hPTMs from breast cancer specimens belonging to different subtypes. The data, which are associated with a recent publication (Pathology tissue-quantitative mass spectrometry analysis to profile histone post-translational modification pat...
Characterization of histones and their post-translational modifications by mass spectrometry
Current Opinion in Chemical Biology, 2007
Histone proteins and their accompanying post-translational modifications have received much attention for their ability to affect chromatin structure and, hence, regulate gene expression. Recently, mass spectrometry has become an important complementary tool for the analysis of histone variants and modification sites, for determining the degree of occupancy of these modifications and for quantifying differential expression of these modifications from various samples. Additionally, as advancements in mass spectrometry technologies continue, the ability to read entire 'histone codes' across large regions of histone polypeptides or intact protein is possible. As chromatin biology demands, mass spectrometry has adapted and continues as a key technology for the analysis of gene regulation networks involving histone modifications.
Quantitative Proteomic Analysis of Histone Modifications
Chemical reviews, 2015
Background: The refractory nature of many cancers remains the main health challenge over the past century. The epigenetic drug, decitabine (DAC), represents one of the most promising therapeutic agents in cancers particularly in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However, its ambiguous anti-tumor mechanism and the unpredictable drug-resistant nature in some population compromise its application in cancer therapy. In crosstalk with DNA methylation, histone post-translational modifications (PTMs) are the key players in modulating the downstream epigenetic status of tumor suppressor genes. This study targets the role of decitabine in epigenetic regulation in leukemia therapy and searches responsive predictors and therapeutic targets for pretreatment evaluation and drug development. Results: A simple, fast, and robust proteomic strategy identified 15 novel PTMs and 60 PTM combinations in two leukemia cell lines (MDS-L and TF-1). Histone modification profiles have been generated and compared between DAC sensitive and resistant groups (n = 3) in response to DAC treatment. Among these histone PTMs, five of which were found differentially upon DAC treatment in drug sensitive and resistant cells: H3.3K36me3, H4K8acK12acK16ac in MDS-L cells; and H3.1K27me1, H3.1K36me1, H3.1K27me1K36me1 in TF-1 cells. They may serve as biomarkers in predicting leukemia and drug responsiveness. In addition, we also explored PTM differences in two cell lines which were developed from early and advanced stages of AML. Three PTMs (H3.1K27me3, H3.1K27me2K36me2 and H3.3K27me2K36me2) are highly abundant in TF-1 cells (advanced AML cell line), suggesting their relevance to leukemogenesis. Our method allowed deep analysis of histone proteins and elucidation of a large number of histone PTMs with high precision and sensitivity. Conclusion: DAC-induced DNA hypomethylation has wide impact on chromatin modifications. This study represents first effort to investigate the undefined epigenetic mechanism of decitabine in leukemia therapy. The identification of 15 novel PTMs and the discovery of several marks have relevance to epigenetic directed therapies.
Nucleic acids research, 2018
Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are inste...
Quantitative proteomic analysis of post-translational modifications of human histones
Molecular & Cellular …, 2006
Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a sitespecific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys 57 from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.