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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 BackgroundHistone 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. ResultsPTMViz 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. ConclusionsHistone 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:
- 10231220
- Publisher / Repository:
- Springer Science + Business Media
- 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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