Mass Spectrometry
Online ISSN : 2186-5116
Print ISSN : 2187-137X
ISSN-L : 2186-5116
Original Article
Mass Spectrometric Characterization of Histone H3 Isolated from in-Vitro Reconstituted and Acetylated Nucleosome Core Particle
Kazumi Saikusa Haruna HidakaShunsuke IzumiSatoko Akashi
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Supplementary material

2020 Volume 9 Issue 1 Pages A0090

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Abstract

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.

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© 2020 Kazumi Saikusa, Haruna Hidaka, Shunsuke Izumi, and Satoko Akashi. This is an open access article distributed under the terms of Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
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