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Abstract Histone post-translational modifications (PTMs) participate in the dynamic regulation of chromatin structure and function, through their chemical nature and specific location within the histone sequence. Alternative analytical approaches for histone PTM studies are required to facilitate the differentiation between ubiquitously present isomers and the detection of low-abundance PTMs Here, we report a high-sensitivity bottom-up method based on nano-liquid chromatography (nLC), trapped ion mobility spectrometry (TIMS), data-dependent acquisition (DDA), parallel accumulation-serial fragmentation (PASEF), and high-resolution time-of-flight tandem mass spectrometry (ToF-MS/MS) for the analysis of histone PTMs. This method was tested in a threatened coral species, the staghorn coral Acropora cervicornis, during an episode of acute thermal stress. The obtained results allowed for the identification of PTM changes in core histones involved in the coral’s heat response. Compared to traditional LC-MS/MS approaches, the incorporation of TIMS and ddaPASEF MS/MS resulted in a highly specific and sensitive method with a wide dynamic range (6 orders of magnitude). This depth of analysis allows for the simultaneous measurement of low-abundance PTM signatures relative to the unmodified form. An added advantage is the ability to mass- and mobility-isolate prior to peptide sequencing, resulting in higher confidence identification of epigenetic markers associated with heat stress in corals (e.g. increased H4 4–17 with 2ac and 3ac, and decreases in H4 4–17 K12ac, K16ac, H4 K20me2, and H2A K5ac, K7ac, K9ac, K12ac, K14ac, and K74ac).
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Direct histone proteoform profiling of the unannotated, endangered coral Acropora cervicornis
Abstract Epigenetic modifications directly regulate the patterns of gene expression by altering DNA accessibility and chromatin structure. A knowledge gap is presented by the need to directly measure these modifications, especially for unannotated organisms with unknown primary histone sequences. In the present work, we developed and applied a novel workflow for identifying and annotating histone proteoforms directly from mass spectrometry-based measurements for the endangered Caribbean coral Acropora cervicornis. Combining high-accuracy de novo top-down and bottom-up analysis based on tandem liquid chromatography, trapped ion mobility spectrometry, non-ergodic electron-based fragmentation, and high-resolution mass spectrometry, near complete primary sequence (up to 99%) and over 86 post-translational modification annotations were obtained from pull-down histone fractions. In the absence of reliable genome annotations, H2A, H2B, and H4 histone sequences and the annotation of the post-translational modifications of the stressed A. cervicornis coral allow for a better understanding of chromatin remodeling and new strategies for targeting intervention and restoration of endangered reef corals.
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- Award ID(s):
- 1921402
- PAR ID:
- 10621571
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 53
- Issue:
- 14
- ISSN:
- 0305-1048
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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