Enhanced Characterization of Histones Using 193 nm Ultraviolet Photodissociation and Proton Transfer Charge Reduction
Enhanced Characterization of Histones Using 193 nm Ultraviolet Photodissociation and Proton Transfer Charge Reduction
复制标题
使用 193 nm 紫外光解和质子转移电荷减少增强组蛋白的表征
DOI：
10.1021/acs.analchem.2c05765
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发表时间：
2023
影响因子：
7.4
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
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作者：
Walker, Jada N.;Lam, Raymond;Brodbelt, Jennifer S.
组蛋白是一系列依赖DNA的过程中的关键参与者，自上而下的表征为研究组蛋白结构和遗传调控机制之间的关系提供了可能性。组蛋白翻译后修饰(PTM)的映射模式需要广泛的骨架裂解来支撑与特定PTM对应的质量移位位置。紫外光解离(UVPD)会导致蛋白质的大量碎裂，这非常适合于PTM定位，但所产生的光谱中充满了碎片离子，这些离子可能具有重叠的同位素分布，从而混淆了去卷积。气相质子转移电荷减少(PTCR)降低了高电荷态离子的电荷态，从而缓解了这种拥堵，并有助于识别额外的序列决定和PTM定域碎片离子。通过将UVPD和PTCR相结合进行组蛋白蛋白质形式分析，小牛胸腺组蛋白H4的序列覆盖率达到91%，其中N端和Lys12处有乙酰化标记，Arg3处有二甲基化标记。UVPD-PTCR在其他组蛋白，如组蛋白H2A的特征方面显示出很大的收益，将单乙酰化的H2A的序列覆盖率从59%提高到77%。
Top-down characterization of histones, proteins that are critical participants in an array of DNA-dependent processes, offers the potential to examine the relationship between histone structure and mechanisms of genetic regulation. Mapping patterns of post-translational modifications (PTMs) of histones requires extensive backbone cleavages to bracket the sites of mass shifts corresponding to specific PTMs. Ultraviolet photodissociation (UVPD) causes substantial fragmentation of proteins, which is well-suited for PTM localization, but the resulting spectra are congested with fragment ions that may have overlapping isotopic distributions that confound deconvolution. Gas-phase proton transfer charge reduction (PTCR) decreases the charge states of highly charged ions, thus alleviating this congestion and facilitating the identification of additional sequence-determining and PTM-localizing fragment ions. By integrating UVPD with PTCR for histone proteoform analyses, sequence coverages up to 91% were achieved for calf thymus histone H4 containing acetylation marks at the N-terminus and Lys12 as well as a dimethylation at Arg3. UVPD-PTCR exhibited large gains in characterization for other histones, such as histone H2A, increasing the sequence coverage from 59 to 77% for monoacetylated H2A.