Efficient protein conformation dynamics characterization enabled by mobility-mass spectrometry.

Protein structure dynamics in solution and from solution to gas phase are important but challenging topics. Great efforts and advances have been made especially since the wide application of ion mobility mass spectrometry (IM-MS), by which protein collision cross section (CCS) in gas phase could be measured. Due to the lack of efficient experimental methods, protein structures in protein databank are typically referred as their structures in solution. Although conventional structural biology techniques provide high-resolution protein structures, complicated and stringent processes also limit their applicability under different solvent conditions, thus preventing the capture of protein dynamics in solution. Enabled by the combination of mobility capillary electrophoresis (MCE) and IM-MS, an efficient experimental protocol was developed to characterize protein conformation dynamics in solution and from solution to gas phase. As a first attempt, key factors that affecting protein conformations were distinguished and evaluated separately, including pH, temperature, softness of ionization process, presence and specific location of disulfide bonds. Although similar extent of unfolding could be observed for different proteins, in-depth analysis reveals that pH decrease from 7.0 to 3.0 dominates the unfolding of proteins without disulfide bonds in conventional ESI-MS experiments; while harshness of the ionization process dominates the unfolding of proteins with disulfide bonds. Second, disulfide bonds show capability of preserving protein conformations in acidic solution environments. However, by monitoring protein conformation dynamics and comparing results from different proteins, it is also found that their capability is position dependent. Surprisingly, disulfide bonds did not show the capability of preserving protein conformations during ionization processes.