Abstract:
Charge reduction in the gas phase provides a direct means of manipulating protein charge state, and when coupled to ion mobility mass spectrometry (IM-MS), it is possible to monitor the effect of charge on protein conformation in the absence of solution. Use of the electron transfer reagent 1,3-dicyanobenzene, coupled with IM-MS, allows us to monitor the effect of charge reduction on the conformation of two proteins deliberately chosen from opposite sides of the order to disorder continuum: bovine pancreatic trypsin inhibitor (BPTI) and beta casein. The ordered BPTI presents compact conformers for each of three charge states accompanied by narrow collision cross-section distributions (TWCCSDN2→He). Upon reduction of BPTI, irrespective of precursor charge state, the TWCCSN2→He decreases to a similar distribution as found for the nESI generated ion of identical charge. The behavior of beta casein upon charge reduction is more complex. It presents over a wide charge state range (9-28), and intermediate charge states (13-18) have broad TWCCSDN2→He with multiple conformations, where both compaction and rearrangement are seen. Further, we see that the TWCCSDN2→He of the latter charge states are even affected by the presence of radical anions. Overall, we conclude that the flexible nature of some proteins result in broad conformational distributions comprised of many families, even for single charge states, and the barrier between different states can be easily overcome by an alteration of the net charge. Graphical Abstract ᅟ.
摘要:
气相中的电荷减少提供了操纵蛋白质电荷状态的直接手段,当耦合到离子迁移质谱(IM-MS)时,在没有溶液的情况下,可以监测电荷对蛋白质构象的影响。使用电子转移试剂1,3-二氰基苯,加上IM-MS,使我们能够监测电荷减少对两种蛋白质构象的影响,这两种蛋白质有意从无序连续体的相反侧选择:牛胰腺胰蛋白酶抑制剂(BPTI)和β酪蛋白。有序的BPTI为三种电荷状态中的每一种都呈现紧凑的构象,并伴有狭窄的碰撞横截面分布(TWCCSDN2→He)。BPTI减少后,不管前体电荷状态如何,TWCCSN2→He降低到与nESI产生的相同电荷的离子相似的分布。β酪蛋白在电荷还原时的行为更复杂。它呈现在一个宽的充电状态范围(9-28),和中间电荷态(13-18)具有宽的TWCCSDN2→He,具有多个构象,在那里看到压实和重排。Further,我们看到后一种电荷态的TWCCSDN2→He甚至受到自由基阴离子存在的影响。总的来说,我们得出的结论是,一些蛋白质的柔性性质导致由许多家族组成的广泛构象分布,即使是单充电状态,并且可以通过改变净电荷来轻松克服不同状态之间的障碍。图形抽象。