Linking Gas-Phase and Solution-Phase Protein Unfolding via Mobile Proton Simulations.

Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase ( in vacuo ) is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between in vacuo and in-solution behavior, we use unbiased molecular dynamics (MD) to create in silico models of in vacuo unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded in silico models for observed charge states of NTL9. The unfolding behavior in silico replicates the behavior in-solution and is in line with the in vacuo observations; however, the theoretical collision cross section (CCS) of the in silico models was lower compared to that of the in vacuo data, which may reflect reduced sampling.