High-Pressure Electrospray Ionization Yields Supercharged Protein Complexes from Native Solutions While Preserving Noncovalent Interactions.

Increasing charge state of protein complexes from native solutions while preserving noncovalent interactions in native mass spectrometry (MS) offers great opportunity to gain deeper insights into gas-phase protein structures. Several previous studies have disclosed the possibility of high pressure in supercharging small proteins, whereas its capability to supercharge large protein assemblies under native conditions and how it might affect protein structures remain open questions. Herein, we demonstrated that the high-pressure-induced supercharging strategy affords unique advantages of supercharging protein complexes with the highest charge state surpassing the Rayleigh limit () and concurrently preserving native-like topology. By examining 32 proteins and protein complexes with molecular weights (MWs) ranging from 8.58 to 801 kDa, we demonstrated that the increased average charge states of macromolecular ions have a strong dependence on the surface areas of native protein conformations and MWs. Factors that might contribute to the high-pressure-induced supercharging capability toward macromolecular ions were discussed. Furthermore, using collision cross section (CCS) variation as a function of charge state, we investigate the effects of gas pressure and charge states on gas-phase structures of proteins and protein complexes. Smaller proteins have the largest CCS variations once supercharged, while macromolecular protein complexes are less affected. The results revealed that both surface density of charge and charged surface basic residues contribute to the observed CCS-charge disciplines for all the macromolecules investigated. Taken together, the results presented here indicate that increasing gas pressure in the ion source affords a rapid, simple, and controlled supercharging method, offering the potency of facilitating further applications of native top-down MS analysis with improved transmission, fragmentation, and detection efficiency.