Departments of Chemistry and Biochemistry, The University of Western Ontario , London, Ontario, N6A 5B7, Canada.
Anal Chem. 2013 Nov 5;85(21):10471-8. doi: 10.1021/ac402490r. Epub 2013 Oct 21.
The coupling of electrospray ionization (ESI) with ion mobility-mass spectrometry (IM-MS) allows structural studies on biological macromolecules in a solvent-free environment. Collision cross sections (CCSs) measured by IM-MS provide a measure of analyte size. For native proteins and their complexes, many structural features can be preserved in the gas phase, making IM-MS a powerful approach for a range of bioanalytical applications. In addition to tightly folded conformers, a large number of partially disordered proteins participate in biological processes and disease mechanisms. It remains unclear to what extent IM-MS is suitable for exploring structural properties of these semifolded species. The current work addresses this question, using myoglobin as model system. This protein follows a sequential unfolding pathway that comprises two partially disordered states, i.e., apo-myoglobin (aMb) at pH 7 and pH 4. IM-MS data acquired for these two conformers were compared to those of native holo-myoglobin (hMb) at pH 7 and extensively unfolded aMb at pH 2. When examining individual aMb charge states, the degree of gas phase unfolding is not strongly correlated with the corresponding solution behavior. A key problem is that non-native conformers generate high ESI charge states, resulting in conformational transitions caused by intramolecular electrostatic repulsion. It is possible to establish a link between solution phase and gas phase structure when normalizing CCS distributions according to their respective ESI-MS signal intensities. This approach yields CCS averages that follow the expected progression hMbpH 7 < aMbpH 7 < aMbpH 4 < aMbpH 2. However, this trend mainly reflects the protonation behavior of the conformers during the ESI process, rather than a genuine memory of solution structure. Overall, our data reveal that electrostatically driven expansion as well as collapse events can lead to disparities between gaseous and solution structures for partially unfolded proteins. IM-MS data on non-native conformers should therefore be interpreted with caution.
电喷雾电离（ESI）与离子淌度-质谱（IM-MS）的联用允许在无溶剂环境中对生物大分子进行结构研究。IM-MS 测量的碰撞截面（CCS）提供了分析物大小的度量。对于天然蛋白质及其复合物，许多结构特征可以在气相中保留，这使得 IM-MS 成为一系列生物分析应用的强大方法。除了紧密折叠的构象体外，大量部分无序的蛋白质参与生物过程和疾病机制。IM-MS 在多大程度上适合探索这些半折叠物种的结构特性仍不清楚。当前的工作使用肌红蛋白作为模型系统来解决这个问题。该蛋白遵循顺序展开途径，包括两个部分无序状态，即 pH 7 时的脱辅基肌红蛋白（apo-myoglobin，aMb）和 pH 4 时的 apo-myoglobin。比较了这两种构象的 IM-MS 数据与 pH 7 时的天然全肌红蛋白（hMb）和 pH 2 时的广泛展开的 apo-myoglobin 的数据。当检查单个 aMb 电荷状态时，气相展开的程度与相应的溶液行为没有很强的相关性。一个关键问题是，非天然构象会产生高 ESI 电荷状态，导致由于分子内静电排斥而产生构象转变。当根据各自的 ESI-MS 信号强度对 CCS 分布进行归一化时，可以在溶液相和气相结构之间建立联系。这种方法得到的 CCS 平均值遵循预期的进展 hMbpH 7 < aMbpH 7 < aMbpH 4 < aMbpH 2。然而，这种趋势主要反映了构象在 ESI 过程中的质子化行为，而不是溶液结构的真实记忆。总的来说，我们的数据表明，静电驱动的扩展和崩溃事件会导致部分展开的蛋白质的气相和溶液结构之间出现差异。因此，应该谨慎解释非天然构象的 IM-MS 数据。
