Faculty of Physics, University of Vienna, Vienna, Austria.
Phys Chem Chem Phys. 2011 Dec 28;13(48):21630-41. doi: 10.1039/c1cp22127b. Epub 2011 Nov 10.
We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase measurements to be correlated to biomolecular structures in solution, low charge state ions should be analyzed. Further, to determine if different solution conditions give rise to ions of different structure, ions of similar charge state should be compared. Non-denatured protein ion densities are found to be in excellent agreement with non-denatured protein ion densities inferred from prior DMA and drift tube measurements made without charge reduction (all ions with densities in the 0.85-1.10 g cm(-3) range), showing that these ions are not strongly influenced by Coulombic stretching nor by analysis method.
我们使用电荷减少电喷雾（ESI）源和随后的离子迁移分析与差分迁移分析仪（DMA，通过法拉第笼静电计和凝聚粒子计数器进行检测）来推断细胞色素 C、溶菌酶、肌红蛋白、卵清蛋白和牛血清白蛋白的单蛋白和多蛋白离子的密度，这些蛋白是由非变性（20 mM 水溶液中的乙酸铵）和变性（1：49.5：49.5，甲酸：甲醇：水）ESI 产生的。电荷减少是通过使用 Po-210 放射性源来实现的，该源会产生大致相等浓度的正离子和负离子。源产生的离子与 ESI 产生的液滴碰撞并减少其电荷，从而防止库仑裂变，与典型的蛋白质 ESI 不同，这导致气相蛋白质离子带有+1 至+3 的多余电荷。因此，电荷减少有助于有效减轻库仑拉伸可能对气相离子结构的任何影响。密度推断是通过确定迁移率直径，并相应地确定球形等效蛋白质体积来实现的。通过这种方法，我们发现对于非变性和变性 ESI 产生的离子，气相蛋白质离子都相对紧凑，其平均密度分别为 0.97 g/cm³和 0.86 g/cm³。与从蛋白质晶体结构预测的相比，非变性 ESI 产生的离子稍紧凑，这表明气相中低电荷状态的蛋白质离子比其溶液构象稍密。虽然在非变性和变性 ESI 产生的离子之间检测到微小差异，但与之前通过漂移管迁移率分析（未采用电荷减少）检测到的离子相比，变性离子的密度要高得多。这表明库仑拉伸通常会导致气相中非紧凑的离子，并表明为了使气相测量与溶液中的生物分子结构相关，应该分析低电荷状态的离子。此外，为了确定不同的溶液条件是否会导致不同结构的离子，应该比较具有相似电荷状态的离子。非变性蛋白质离子密度与先前没有电荷减少的 DMA 和漂移管测量推断的非变性蛋白质离子密度（密度均在 0.85-1.10 g/cm³范围内的所有离子）非常吻合，表明这些离子不受库仑拉伸的强烈影响，也不受分析方法的影响。
