Native Mass Spectrometry of Protein and DNA Complexes Prepared in Nonvolatile Buffers
Native Mass Spectrometry of Protein and DNA Complexes Prepared in Nonvolatile Buffers
复制标题
DOI：
10.1021/jasms.9b00145
复制
发表时间：
2020-03-01
影响因子：
3.2
通讯作者：
Akashi, Satoko
中科院分区：
文献类型：
--
作者：
Saikusa, Kazumi;Kato, Daiki;Akashi, Satoko
无机盐和非挥发性缓冲液成分会影响蛋白质的结构和稳定性，如果没有它们，一些蛋白质复合物就无法维持其功能和结构。然而，众所周知，这些成分会在电喷雾电离过程中抑制分析物电离。因此，为了建立在存在非挥发性缓冲液成分的情况下通过天然质谱（天然 MS）观察蛋白质和 DNA 复合物的完整离子的适当方法，我们在此检查了添加乙酸铵到模型同四聚体蛋白乙醇脱氢酶（ADH）的效果，该模型是在一系列非挥发性缓冲液中制备的，包括 Tris-HCl、磷酸盐和 HEPES 缓冲液。此外，还检查了核小体核心颗粒 (NCP)（一种在非挥发性缓冲液中制备的大型蛋白质 DNA 复合物）的天然 MS。添加乙酸铵后可以观察到完整的 ADH 和 NCP 离子，但 NCP 不需要像 ADH 那样高浓度的乙酸铵。对于在 Tris-HCl 中添加比 ADH 更少量的乙酸铵制备的 NCP，可以观察到具有不同电荷数的清晰峰。这表明添加剂对生物分子复合物的天然 MS 的影响可能会根据存在的分子内相互作用而变化。更具体地说，NCP 主要通过静电相互作用来稳定，而 ADH 四聚体则取决于四个亚基之间疏水相互作用的存在。因此，本文提出的结果有望有助于转录过程中瞬时形成的不稳定蛋白质-DNA 复合物的结构生物学研究。
Inorganic salts and nonvolatile-buffer components affect the structure and stability of proteins, and some protein complexes are unable to maintain their function and structure without them. However, it is well-known that these components cause suppression of analyte ionization during the electrospray ionization process. Thus, to establish appropriate methods for observation of the intact ions of protein and DNA complexes by native mass spectrometry (native MS) in the presence of nonvolatile buffer components, we herein examined the effect of ammonium acetate addition to a model homotetramer protein, alcohol dehydrogenase (ADH), which was prepared in a range of nonvolatile buffers, including Tris-HCl, phosphate, and HEPES buffers. Furthermore, native MS of nucleosome core particle (NCP), a large protein DNA complex, prepared in nonvolatile buffer, was also examined. Intact ADH and NCP ions could be observed upon the addition of ammonium acetate, but NCP does not require as high of a concentration of ammonium acetate as ADH. Well-resolved peaks with different charge numbers could be observed for NCP prepared in Tris-HCl by addition of a lower amount of ammonium acetate than for ADH. This suggests that the effects of additives on native MS of biomolecular complexes can vary depending on the intramolecular interactions present. More specifically, NCP is stabilized mainly by electrostatic interactions, whereas the ADH tetramer depends on the presence of hydrophobic interactions between the four subunits. The results presented herein therefore are expected to contribute to structural biology studies of unstable protein-DNA complexes that are formed transiently during the transcription process.