Chernogolovka Branch of the N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, Chernogolovka, Moscow, 142432, Russia.
Federal Research Center of Problems of Chemical Physics and Medicinal Chemistry, Russian Academy of Sciences, Chernogolovka, Moscow, 142432, Russia.
Anal Bioanal Chem. 2023 May;415(12):2193-2207. doi: 10.1007/s00216-023-04625-7. Epub 2023 Mar 21.
The work is dedicated to further development of our described method for analyzing mass spectra of biomolecules acquired as a result of hydrogen-deuterium exchange reactions (HDXs). The modified method consists of separating HDX distributions via their approximations by a minimum number of components corresponding to independent H/D substitutions and independent charge carrier retentions in different spatial isoforms or conformations of biomolecules with unknown primary structures. In this case, neither the natural isotopic distribution nor the exact number of active sites involved in HDXs and H or D attachments can be determined in advance. Original H/D electrospray mass spectra of an apamin solution were taken from our previous work. In that work, taking into account the natural isotopic distribution of apamin molecules, three main conformations of apamin ions were found as a result of separating the H/D mass spectra of the apamin solution for the gas flow with the addition of about 10% ND molecules. Using the proposed modified method that does not require knowledge of the primary structure of the biomolecules gave similar results with slight deviations of calculated HDX distributions of the apamin ions from those obtained earlier. The maximum difference between mean values of the calculated HDX distributions for ions of the same charge in both cases does not exceed a few percent. In addition, HDX mass spectra of the apamin complex with an adduct of unknown structure were processed. Such analysis gave also three main fractions of ions with relatively large contributions when ND was injected into a radio-frequency quadrupole. In the absence of ND flow, the results of calculations for apamin and its complex were close to each other too. The formation of the apamin complex most probably in solution was confirmed by performed calculations.
这项工作致力于进一步发展我们描述的用于分析作为氢氘交换反应（HDX）结果获得的生物分子质谱的方法。该改进方法包括通过用与生物分子的不同空间构象或构象中独立的 H/D 取代和独立的电荷载体保留相对应的最小数量的组分来分离 HDX 分布，而这些构象或构象的一级结构未知。在这种情况下，既不能事先确定天然同位素分布，也不能确定涉及 HDX 和 H 或 D 附着的活性位点的确切数量。来自我们以前工作的一种 Apamin 溶液的原始 H/D 电喷雾质谱被采用。在该工作中，考虑到 Apamin 分子的天然同位素分布，通过分离 Apamin 溶液的 H/D 质谱，在添加约 10% ND 分子的气流下，发现了 Apamin 离子的三种主要构象。使用不需要生物分子一级结构知识的提议的改进方法给出了类似的结果，Apamin 离子的计算 HDX 分布与先前获得的结果略有偏差。在这两种情况下，相同电荷的离子的计算 HDX 分布的平均值之间的最大差异不超过几个百分点。此外，处理了与未知结构的加合物的 Apamin 复合物的 HDX 质谱。当将 ND 注入射频四极时，这种分析也给出了具有相对较大贡献的三个主要离子分数。在没有 ND 流的情况下，Apamin 及其复合物的计算结果也非常接近。通过进行的计算证实了 Apamin 复合物很可能在溶液中形成。
