Collisional and Coulombic Unfolding of Gas-Phase Proteins: High Correlation to Their Domain Structures in Solution

The three‐dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higher‐order assemblies. While important coursed‐grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas‐phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility‐mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8–78 kDa. This correlation and its potential uses for structural biology is discussed. Gas‐phase unfolding is used as a means to determine the number of autonomously folded domains within monomeric proteins. Ion‐mobility mass spectrometry data show a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8–78 kDa. CCS=collision cross‐section.