Brundridge Nicole M, Koers Alexander M, McLuckey Scott A
Department of Chemistry, Purdue University 560 Oval Drive, West Lafayette, Indiana 47907, United States.
J Am Soc Mass Spectrom. 2024 Jun 5;35(6):1342-1351. doi: 10.1021/jasms.4c00099. Epub 2024 May 22.
The final stages of the charged residue mechanism/model (CRM) for ion generation via electrospray ionization (ESI) involves the binding of excess charge onto analyte species. Ions of both polarities can bind to the analyte with an excess of ions of the same polarity as the droplet. For large biomolecule/biocomplex ions, which are commonly the species of interest in native mass spectrometry (MS), the binding of acids and salts onto the analyte can lead to extensive broadening of ion signals due to adduction. Therefore, heating step(s) to facilitate desolvation and salt adduct removal are commonplace. In this work, we describe an approach to study the final stages of CRM using gas-phase ion/ion reactions to generate analyte ion/salt clusters of well-defined composition, followed by gas-phase collision-induced dissociation (CID). While there are many variables that can be studied systematically via this approach, the work described herein is focused on salt clusters of the form [NaX], where X = acetate (Ac), chloride (Cl), or nitrate (NO), in reaction with a common charge state of ubiquitin as well as several model peptides. Experiments in which equimolar quantities of each salt (i.e., NaAc, NaCl, and NaNO) are subjected to ESI with ubiquitin (Ubi) and gas-phase ion/ion reaction studies involving [NaX] and [Ubi + 6H] show similar trends, in terms of the extent of sodium ion incorporation into the protein ions. Ion/ion reaction studies using model peptides show that the acetate-containing salt transfers significantly more Na ions into the peptide ions. Exchange of Na for H is shown to occur at the C-terminus and at up to all of the amide linkages using [NaX], whereas only the C-terminus engages in Na/H exchange with [NaCl] and [Na(NO)]. In the latter cases, an additional Na is taken up as the excess positive charge, presumably due to solvation of the charge by multiple sites (e.g., carbonyl oxygens and basic sites).
通过电喷雾电离（ESI）产生离子的带电残基机制/模型（CRM）的最后阶段涉及将多余电荷结合到分析物物种上。两种极性的离子都可以与分析物结合，且与液滴具有相同极性的离子过量。对于大型生物分子/生物复合物离子，它们通常是天然质谱（MS）中感兴趣的物种，酸和盐与分析物的结合会由于加合作用导致离子信号大幅展宽。因此，用于促进去溶剂化和去除盐加合物的加热步骤很常见。在这项工作中，我们描述了一种研究CRM最后阶段的方法，即利用气相离子/离子反应生成组成明确的分析物离子/盐簇，随后进行气相碰撞诱导解离（CID）。虽然通过这种方法可以系统地研究许多变量，但本文所述工作聚焦于[NaX]形式的盐簇，其中X = 乙酸根（Ac）、氯离子（Cl）或硝酸根（NO），与泛素的常见电荷态以及几种模型肽发生反应。将等摩尔量的每种盐（即NaAc、NaCl和NaNO）与泛素（Ubi）进行ESI实验，以及涉及[NaX]和[Ubi + 6H]的气相离子/离子反应研究表明，就钠离子掺入蛋白质离子的程度而言，呈现相似趋势。使用模型肽的离子/离子反应研究表明，含乙酸根的盐向肽离子中转移的钠离子明显更多。使用[NaX]时，Na与H的交换发生在C端以及多达所有的酰胺键处，而使用[NaCl]和[Na(NO)]时，只有C端参与Na/H交换。在后一种情况下，会额外吸收一个Na作为多余的正电荷，推测是由于电荷被多个位点（如羰基氧和碱性位点）溶剂化。
