Department of Mechanical Engineering, Yale University, USA.
Phys Chem Chem Phys. 2010 Nov 7;12(41):13476-83. doi: 10.1039/c0cp01208d. Epub 2010 Sep 28.
We investigate whether "supercharging" reagents able to shift the charge state distributions (CSDs) of electrosprayed protein ions upward also influence gas-phase protein structure. A differential mobility analyzer and a mass spectrometer are combined in series (DMA-MS) to measure the mass and mobility of monomer and multimeric phosphorylase B ions (monomer molecular weight ∼97 kDa) in atmospheric pressure air. Proteins are electrosprayed from charge-reducing triethylammonium formate in water (pH = 6.8) with and without the addition of the supercharging reagent tetramethylene sulfone (sulfolane). Because the DMA measures ion mobility prior to collisional heating or declustering, it probes the structure of supercharged protein ions immediately following solvent (water) evaporation. As in prior studies, the addition of sulfolane is found to drastically increase both the mean and maximum charge state of phosphorylase B ions. Ions from all protein n-mers were found to yield mobilities that, for a given charge state, were ∼6-10% higher in the absence of sulfolane. We find that the mobility decrease which arises with sulfolane is substantially smaller than that typically observed for folded-to-unfolded transitions in protein ions (where a ∼60% decrease in mobility is typical), suggesting that supercharging reagents do not cause structural protein modifications in solution as large as noted recently by Williams and colleagues [E. R. Williams et al., J. Am. Soc. Mass Spectrom., 2010, 21, 1762-1774]. In fact, the measurements described here indicate that the modest mobility decrease observed can be partly attributed to sulfolane trapping within the protein ions during DMA measurements, and probably also in solution. As the most abundant peaks in measured mass-mobility spectra for ions produced with and without sulfolane correspond to non-covalently bound phosphorylase B dimers, we find that in spite of a change in mobility/cross section, sulfolane addition does not substantially alter the structure of non-covalently bound protein complexes in the gas-phase.
我们研究了“增强”试剂是否能够改变电喷雾蛋白质离子的电荷状态分布（CSD），同时也会影响蛋白质的气相结构。我们将差分迁移分析仪和质谱仪串联（DMA-MS），以测量大气压空气中单体和多聚体磷酸化酶 B 离子（单体分子量约为 97 kDa）的质量和迁移率。我们将蛋白质从带负电荷的甲酸三乙铵在水中（pH = 6.8）中电喷雾，同时加入和不加入超荷试剂四亚甲基砜（环丁砜）。由于 DMA 在碰撞加热或解簇之前测量离子迁移率，因此它可以在溶剂（水）蒸发后立即探测超荷蛋白质离子的结构。与之前的研究一样，我们发现添加四亚甲基砜会极大地增加磷酸化酶 B 离子的平均电荷状态和最大电荷状态。我们发现，对于给定的电荷状态，在没有四亚甲基砜的情况下，所有蛋白质 n-聚体的离子迁移率都提高了约 6-10%。我们发现，与折叠到未折叠转变中典型的蛋白质离子相比，四亚甲基砜引起的迁移率下降要小得多（通常为 60%），这表明超荷试剂不会像最近 Williams 等人[E. R. Williams 等人，J. Am. Soc. Mass Spectrom.，2010，21，1762-1774]所指出的那样，在溶液中引起蛋白质结构的明显变化。事实上，这里描述的测量结果表明，观察到的适度迁移率下降部分归因于 DMA 测量过程中四亚甲基砜在蛋白质离子内的捕获，而且可能也在溶液中。由于在有和没有四亚甲基砜的情况下产生的离子的测量质量-迁移率谱中最丰富的峰对应于非共价结合的磷酸化酶 B 二聚体，我们发现尽管迁移率/横截面积发生了变化，但四亚甲基砜的添加并没有实质性地改变气相中非共价结合蛋白质复合物的结构。
