- 著者
- Nanako Azegami Rina Taguchi Noa Suzuki Yusuke Sakata Tsuyoshi Konuma Satoko Akashi
- 出版者
- The Mass Spectrometry Society of Japan
- 雑誌
- Mass Spectrometry (ISSN:2187137X)
- 巻号頁・発行日
- vol.11, no.1, pp.A0110, 2022-12-28 (Released:2022-12-28)
- 参考文献数
- 35
The contribution of disordered regions to protein function and structure is a relatively new field of study and of particular significance as their function has been implicated in some human diseases. Our objective was to analyze various deletion mutants of the bromodomain-containing protein 4 (BRD4) using native mass spectrometry to characterize the gas-phase behavior of the disordered region connected to the folded domain. A protein with a single bromodomain but no long disordered linker displayed a narrow charge distribution at low charge states, suggesting a compact structure. In contrast, proteins containing one or two bromodomains connected to a long disordered region exhibited multimodal charge distributions, suggesting the presence of compact and elongated conformers. In the presence of a pan-BET-bromodomain inhibitor, JQ1, the protein–JQ1 complex ions had relatively small numbers of positive charges, corresponding to compact conformers. In contrast, the ions with extremely high charge states did not form a complex with JQ1. This suggests that all of the JQ1-bound BRD4 proteins in the gas phase are in a compact conformation, including the linker region, while the unbound forms are considerably elongated. Although these are gas-phase phenomena, it is possible that the long disordered linker connected to the bromodomain causes the denaturation of the folded domain, which, in turn, affects its JQ1 recognition.
- 著者
- Kazumi Saikusa Haruna Hidaka Shunsuke Izumi Satoko Akashi
- 出版者
- The Mass Spectrometry Society of Japan
- 雑誌
- Mass Spectrometry (ISSN:2187137X)
- 巻号頁・発行日
- vol.9, no.1, pp.A0090, 2020-10-31 (Released:2020-10-31)
- 参考文献数
- 30
- 被引用文献数
- 1
Post-translational modifications (PTMs) of histone N-terminal tails in nucleosome core particle (NCP), such as acetylation, play crucial roles in regulating gene expression. To unveil the regulation mechanism, atomic-level structural analysis of in-vitro modified NCP is effective with verifying the PTMs of histones. So far, identification of PTMs of NCP originating from living cells has mainly been performed using mass spectrometry (MS) techniques, such as bottom-up approach. The bottom-up approach is the most established method for protein characterization, but it does not always provide sufficient information on the acetylated sites of lysine residues in the histone tails if trypsin digestion is carried out. For histone proteins, which have many basic amino acids, trypsin generates too many short fragments that cannot be perfectly analyzed by tandem MS. In this study, we investigated the in vitro acetylation sites in the histone H3 tail using a top-down sequence analysis, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiment, in combination with aminopeptidase digestion. Aminopeptidase can cleave peptide bonds one-by-one from the N-terminus of peptides or proteins, generating N-terminally truncated peptides and/or proteins. As a result, it was identified that this method enables sequence characterization of the entire region of the H3 tail. Also, application of this method to H3 in in-vitro acetylated NCP enabled assigning acetylation sites of H3. Thus, this method was found to be effective for obtaining information on in-vitro acetylation of NCP for structural biology study.