Jia Mengxuan, Song Yang, Du Chen, Wysocki Vicki H
The Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States.
Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, Ohio 43210, United States.
J Am Soc Mass Spectrom. 2023 Oct 4;34(10):2166-2175. doi: 10.1021/jasms.3c00142. Epub 2023 Aug 17.
Charge partitioning during the dissociation of protein complexes in the gas phase is influenced by many factors, such as interfacial interactions, protein flexibility, protein conformation, and dissociation methods. In the present work, two cysteine-containing homodimer proteins, β-lactoglobulin and α-lactalbumin, with the disulfide bonds intact and reduced, were used to gain insight into the charge partitioning behaviors of collision-induced dissociation (CID) and surface-induced dissociation (SID) processes. For these proteins, we find that restructuring dominates with CID and dissociation with symmetric charge partitioning dominates with SID, regardless of whether intramolecular disulfide bonds are oxidized or reduced. CID of the charge-reduced dimeric protein complex leads to a precursor with a slightly smaller collision cross section (CCS), greater stability, and more symmetrically distributed charges than the significantly expanded form produced by CID of the higher charged dimer. Collision-induced unfolding plots demonstrate that the unfolding-restructuring of the protein complexes initiates the charge migration of higher charge-state precursors. Overall, gas collisions reveal the charge-dependent restructuring/unfolding properties of the protein precursor, while surface collisions lead predominantly to more charge-symmetric monomer separation. CID's multiple low-energy collisions sequentially reorganize intra- and intermolecular bonds, while SID's large-step energy jump cleaves intermolecular interfacial bonds in preference to reorganizing intramolecular bonds. The activated population of precursors that have taken on energy without dissociating (populated in CID over a wide range of collision energies, populated in SID for only a narrow distribution of collision energies near the onset of dissociation) is expected to be restructured, regardless of the activation method.
气相中蛋白质复合物解离过程中的电荷分配受许多因素影响，如界面相互作用、蛋白质柔韧性、蛋白质构象和解离方法。在本研究中，使用了两种含半胱氨酸的同二聚体蛋白，即二硫键完整和还原的β-乳球蛋白和α-乳白蛋白，以深入了解碰撞诱导解离（CID）和表面诱导解离（SID）过程中的电荷分配行为。对于这些蛋白质，我们发现，无论分子内二硫键是氧化还是还原，CID时重组占主导，而SID时对称电荷分配的解离占主导。电荷减少的二聚体蛋白复合物的CID导致一种前体，其碰撞截面（CCS）略小、稳定性更高且电荷分布比高电荷二聚体的CID产生的显著扩展形式更对称。碰撞诱导的去折叠图表明，蛋白质复合物的去折叠-重组引发了高电荷态前体的电荷迁移。总体而言，气体碰撞揭示了蛋白质前体的电荷依赖性重组/去折叠特性，而表面碰撞主要导致电荷更对称的单体分离。CID的多次低能碰撞依次重组分子内和分子间键，而SID的大步能量跃迁优先裂解分子间界面键而非重组分子内键。无论激活方法如何，已吸收能量但未解离的前体的活化群体（在CID的广泛碰撞能量范围内存在，在SID中仅在解离开始附近的窄碰撞能量分布中存在）预计会发生重组。
