Saikusa Kazumi, Hidaka Haruna, Izumi Shunsuke, Akashi Satoko
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8563, Japan.
Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
Mass Spectrom (Tokyo). 2020;9(1):A0090. doi: 10.5702/massspectrometry.A0090. Epub 2020 Oct 31.
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 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 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 acetylated NCP enabled assigning acetylation sites of H3. Thus, this method was found to be effective for obtaining information on acetylation of NCP for structural biology study.
核小体核心颗粒（NCP）中组蛋白N端尾巴的翻译后修饰（PTM），如乙酰化，在调节基因表达中起关键作用。为了揭示调控机制，对修饰的NCP进行原子水平的结构分析对于验证组蛋白的PTM是有效的。到目前为止，源自活细胞的NCP的PTM鉴定主要使用质谱（MS）技术，如自下而上的方法。自下而上的方法是蛋白质表征中最成熟的方法，但如果进行胰蛋白酶消化，它并不总是能提供关于组蛋白尾巴中赖氨酸残基乙酰化位点的足够信息。对于含有许多碱性氨基酸的组蛋白，胰蛋白酶会产生太多短片段，无法通过串联质谱进行完美分析。在本研究中，我们结合氨肽酶消化，使用自上而下的序列分析、基质辅助激光解吸/电离源内衰变（MALDI-ISD）实验研究了组蛋白H3尾巴中的乙酰化位点。氨肽酶可以从肽或蛋白质的N端逐个切割肽键，产生N端截短的肽和/或蛋白质。结果表明，该方法能够对H3尾巴的整个区域进行序列表征。此外，将该方法应用于乙酰化NCP中的H3，能够确定H3的乙酰化位点。因此，发现该方法对于获得用于结构生物学研究的NCP乙酰化信息是有效的。
