Distinguishing Protein Chemical Topologies Using Supercharging Ion Mobility Spectrometry‐Mass Spectrometry

A technique combining ion mobility spectrometry‐mass spectrometry (IMS‐MS) and supercharging electrospray ionization (ESI) has been demonstrated to differentiate protein chemical topology effectively. Incorporating as many charges as possible into proteins via supercharging ESI allows the protein chains to be largely unfolded and stretched, revealing their hidden chemical topology. Different chemical topologies result in differing geometrical sizes of the unfolded proteins due to constraints in torsional rotations in cyclic domains. By introducing new topological indices, such as the chain‐length‐normalized collision cross‐section (CCS) and the maximum charge state (zM) in the extensively unfolded state, we were able to successfully differentiate various protein chemical topologies, including linear chains, ring‐containing topologies (lasso, tadpole, multicyclics, etc.), and mechanically interlocked rings, like catenanes. The topological features of complex proteins, previously concealed by their inherent complexity, can now be easily classified through the application of supercharging electrospray ionization coupled with ion mobility spectrometry‐mass spectrometry. By overcharging the protein backbones, limitations in protein elongation depending on the protein topology become evident, thereby establishing a visual standard for discerning protein topologies.