Partially disordered proteins studied by ion mobility-mass spectrometry: implications for the preservation of...

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.