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Abstract Mass spectrometry (MS)‐based top‐down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post‐translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high‐resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS‐PAGE‐based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI‐MS) with capillary zone electrophoresis (CZE)‐MS/MS for high‐resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS‐PAGE gel and follow‐up cleanup as well as CZE‐MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high‐resolution separation and characterization of histone proteoforms. SDS‐PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high‐resolution separations of SDS‐PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.
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PTMViz: a tool for analyzing and visualizing histone post translational modification data
Abstract Background Histone post-translational modifications (PTMs) play an important role in our system by regulating the structure of chromatin and therefore contribute to the regulation of gene and protein expression. Irregularities in histone PTMs can lead to a variety of different diseases including various forms of cancer. Histone modifications are analyzed using high resolution mass spectrometry, which generate large amounts of data that requires sophisticated bioinformatics tools for analysis and visualization. PTMViz is designed for downstream differential abundance analysis and visualization of both protein and/or histone modifications. Results PTMViz provides users with data tables and visualization plots of significantly differentiated proteins and histone PTMs between two sample groups. All the data is packaged into interactive data tables and graphs using the Shiny platform to help the user explore the results in a fast and efficient manner to assess if changes in the system are due to protein abundance changes or epigenetic changes. In the example data provided, we identified several proteins differentially regulated in the dopaminergic pathway between mice treated with methamphetamine compared to a saline control. We also identified histone post-translational modifications including histone H3K9me, H3K27me3, H4K16ac, and that were regulated due to drug exposure. Conclusions Histone modifications play an integral role in the regulation of gene expression. PTMViz provides an interactive platform for analyzing proteins and histone post-translational modifications from mass spectrometry data in order to quickly identify differentially expressed proteins and PTMs.
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- Award ID(s):
- 1946391
- PAR ID:
- 10321618
- Date Published:
- Journal Name:
- BMC Bioinformatics
- Volume:
- 22
- Issue:
- 1
- ISSN:
- 1471-2105
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Histone post-translational modifications (PTMs) are epigenetic marks that play a critical role in the expression and maintenance of DNA, but they remain largely uninvestigated in non-model organisms due to technical challenges. To begin alleviating this issue, we developed a workflow for histone PTM analysis in the Mozambique tilapia (Oreochromis mossambicus), being a widespread and environmentally hardy fish, using mass spectrometry methods. By incorporating multiple protein digestion methods into the preparation of each sample, we reliably quantified 503 biologically relevant histone PTMs. All of these histone PTMs, collectively referred to as the global histone PTM landscape, were characterized in the gills, kidney, and testes of this fish. By comparing the global histone PTM landscape between the three tissues, we found that 90.46% of histone PTMs were tissue-dependent. The workflow and tools for histone PTM analysis described in this study are now publicly available and enable comprehensive investigation into the influence of environmental stress on histone PTMs in non-model organisms. Given the functionality and flexibility of histone PTMs, we anticipate that the study of histone PTMs in ecologically relevant contexts will provide ground-breaking insights into comparative physiology and evolution.more » « less
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Histone post-translational modifications (PTMs) are epigenetic marks that operate within the central dogma of molecular biology: upon an environmental stimulus, the histone PTMs surrounding DNA can be changed in a way that modifies gene expression, and, therefore, the abundance and composition of RNA and proteins within cells (1). Once a change is induced, histone PTMs can offer organisms resilience to their environments through processes such as developmental plasticity (2, 3). The purpose of this study was to investigate whether histone PTMs mediate developmental plasticity in Mozambique tilapia facing salinity challenges. To this aim, we exposed fish to either freshwater or hypersalinity during their early critical window of development, then continued to raise the fish in either freshwater or seawater, respectively, for 18 months. Once the fish reached adulthood, we acclimated them to either freshwater or seawater. Following salinity treatments, we quantified 343 histone PTMs in the gills of each fish. We show here that histone PTMs differ dramatically between fish exposed to distinct environmental conditions for 18 months, and that the majority of histone PTM alterations persist for at least four weeks. However, histone PTMs respond minimally to salinity acclimation during adulthood. These results challenge our prior assumptions regarding the timescale of the histone PTM response, demonstrating that it does not necessarily precede the proteomic response or acclimation. Although this finding complicates our interpretation of developmental plasticity, it signifies that histone PTMs reflect prolonged exposure to environmental conditions.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1186/s12859-021-04166-9
