Recent advances in native mass spectrometry have enabled its use to probe the structure of and interactions within biomolecular complexes. Surface-induced dissociation, in which inter- and intramolecular interactions are disrupted following an energetic ion-surface collision, is a method that can directly interrogate the topology of protein complexes. However, a quantitative relationship between the ion kinetic energy at the moment of surface collision and the internal energy deposited into the ion has not yet been established for proteins. The factors affecting energy deposition in surface-induced unfolding (SIU) of protein monomers were investigated and a calibration relating laboratory-frame kinetic energy to internal energy developed. Protein monomers were unfolded by SIU and by collision-induced unfolding (CIU). CIU and SIU cause proteins to undergo the same unfolding transitions at different values of laboratory-frame kinetic energy. There is a strong correlation between the SIU and CIU energies, demonstrating that SIU, like CIU, can largely be understood as a thermal process. The change in internal energy in CIU was modeled using a Monte Carlo approach and theory. Computed values of the overall efficiency were found to be approximately 25% and used to rescale the CIU energy axis and relate nominal SIU energies to internal energy. The energy deposition efficiency in SIU increases with mass and kinetic energy from a low of ∼20% to a high of ∼68%, indicating that the effective mass of the surface increases along with the mass of the ion. The effect of ion structure on energy deposition was probed using multiple stages of ion activation. Energy deposition in SIU strongly depends on structure, decreasing as the protein is elongated, due to decreased effective protein-surface collisional cross section and increased transfer to rotational modes.
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