Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, United States.
J Am Soc Mass Spectrom. 2024 Sep 4;35(9):2237-2247. doi: 10.1021/jasms.4c00292. Epub 2024 Aug 19.
Multiply charged ions produced by electrospray ionization (ESI) of heterogeneous mixtures of macromolecular analytes under native conditions are typically confined to relatively narrow ranges of mass-to-charge (/) ratio, often with extensive overlap. This scenario makes charge and mass assignments extremely challenging, particularly when individual charge states are unresolved. An ion/ion reaction strategy involving multiply charged ion attachment (MIA) to the mixture components in a narrow range of / can facilitate charge and mass assignment. In MIA operation, multiply charged reagent ions are attached to the analyte ions of opposite polarity to provide large / displacements resulting from both large changes in mass and charge. However, charge reduction of the high / ions initially generated under native ESI conditions requires the ability to isolate high / ions and to analyze even higher / product ions. Digital ion trap (DIT) operation offers means for both high / ion isolation and high / mass analysis, in addition to providing conditions for the reaction of oppositely charged ions. The feasibility of conducting MIA experiments in a DIT that takes advantage of high / ion operation is demonstrated here using a tandem 2D-3D DIT instrument. Proof-of-concept MIA experiments with cations derived from β-galactosidase using the 20- charge state of human serum immunoglobulin G (IgG, ∼149 kDa) as the reagent anion are described. MIA experiments involving mixtures of ions derived from the ribosome are also described. For example, three components in a mixture of 70S particles (>2.2 MDa) were resolved and assigned with masses and charges following an MIA experiment involving the 20- charge state of human serum IgG.
在天然条件下，通过电喷雾电离（ESI）对大分子分析物的多电荷离子进行异质混合物的产生通常局限于质量电荷比（/）的相对较窄范围，通常存在广泛的重叠。这种情况使得电荷和质量分配变得极具挑战性，特别是当单个电荷状态无法分辨时。涉及多电荷离子附着（MIA）到混合物成分的离子/离子反应策略在/的窄范围内可以促进电荷和质量分配。在 MIA 操作中，多电荷试剂离子与相反极性的分析物离子附着，以提供大/位移，这是由于质量和电荷的巨大变化。然而，在天然 ESI 条件下最初产生的高/离子的电荷减少需要能够隔离高/离子并分析甚至更高/的产物离子的能力。数字离子阱（DIT）操作除了为相反电荷离子的反应提供条件外，还提供了高/离子隔离和高/质量分析的手段。这里利用串联二维-三维 DIT 仪器展示了在利用高/离子操作的 DIT 中进行 MIA 实验的可行性。使用来自人血清免疫球蛋白 G（IgG，~149 kDa）的 20 电荷状态作为试剂阴离子的β-半乳糖苷酶衍生阳离子的概念验证 MIA 实验进行了描述。还描述了涉及核糖体衍生离子混合物的 MIA 实验。例如，在涉及 70S 颗粒（>2.2 MDa）的混合物的 MIA 实验之后，混合物中的三个成分被分离并分配了质量和电荷。
