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. 2025 Jul 19;53(14):gkaf740.
doi: 10.1093/nar/gkaf740.

Direct histone proteoform profiling of the unannotated, endangered coral Acropora cervicornis

Cassandra N Fuller  1 , Sabrina Mansoor  2 , Kevin Jeanne Dit Fouque  1 , Lilian Valadares Tose  1 , Javier Rodriguez-Casariego  2   3 , Mariangela Kosmopoulou  4 , Detlev Suckau  5 , Francisca N de Luna Vitorino  6 , Benjamin A Garcia  6 , Jose M Eirin-Lopez  2 , Francisco Fernandez-Lima  1   7
Affiliations

Direct histone proteoform profiling of the unannotated, endangered coral Acropora cervicornis

Cassandra N Fuller et al. Nucleic Acids Res. .

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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Conflict of interest statement

The authors declare the following competing financial interest(s): M.K. and D.S. are employees of Bruker Daltonics, manufacturer of the timsTOF Pro 2 and developer of the OSc software.

Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Direct protein sequence generation and proteoform PTM annotation based on liquid chromatography (online and offline), TIMS, ECD fragmentation, and high-resolution MS for A. cervicornis. (A) Histone extraction from A. cervicornis coral. (B) Top-down identification of histone variants with subsequent ECD-MS/MS and de novo sequencing. (C) Bottom-up nLC-TIMS-MS/MS peptide confirmation.
Figure 2.
Figure 2.
MS-BLAST results for A. cervicornis 32.0 and 36.9 min fractions. (A) A. cervicornis H4 and (B) H4.S top 9 matching proteins found in the SwissProt database with total scores and mapped protein segments, and proposed sequences for each fraction. The single amino acid variation A83S is highlighted.
Figure 3.
Figure 3.
Acropora cervicornis histone H4 detected variants and corresponding proteoforms. (A) H4 and (B) H4.S variants with PTM positions shown with specific PTM combinations below (dotted lines; left). The deconvoluted MS1 spectra, labeled with mass shifts corresponding to the observed PTM(s) (branched lines, colored according to the PTM represented by the observed mass shift from the unmodified mass), and bar plot (right), showing the relative abundances of each of the observed proteoforms with numbered labels corresponding to the PTM combinations shown (dotted lines). Details on sequence coverage at the protein and peptide levels are provided in supplementary data.
Figure 4.
Figure 4.
Acropora cervicornis histone H2A and H2B detected variants and corresponding proteoforms. (A) H2A & H2A.A, (B) H2B-1, (C) H2B-2 & H2B-2K, and (D) H2B-3 variants with PTM positions shown with specific PTM combinations below (dotted lines; left). The deconvoluted MS1 spectra, labeled with mass shifts corresponding to the observed PTM(s) (branched lines, colored according to the PTM represented by the observed mass shift from the unmodified mass), and bar plot (right), showing the relative abundances of each of the observed proteoforms with numbered labels corresponding to the PTM combinations shown (dotted lines). Details on sequence coverage at the protein and peptide levels are provided in supplementary data.
Figure 5.
Figure 5.
Bottom-up confirmation of histone peptides using nLC-TIMS-PASEF-ToF MS/MS. Detected peptides for H4, H2A, and H2B variants with relative abundances.
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