Protein Stability in Solution and in the Gas Phase.
Abstract
Electrospray Ionization mass spectrometry (ESI-MS) is widely used for probing proteins, yet many aspects of this technique remain elusive. Using MS, ion mobility spectrometry (IMS), and circular dichroism (CD) spectroscopy, this thesis sheds light on the stability differences of proteins in the gas phase and solution. After a general introduction (Chapter 1), Chapter 2 scrutinizes some aspects of native ESI. Our data highlight the significance of cone voltage in maintaining a native-like fold and show the advantage of using NH4Ac in protein experiments. Chapter 3 focuses on hydrogen/deuterium exchange (HDX)-MS. Several studies have reported that D2O enhances the stability of proteins. We corroborated this effect through thermal unfolding assays. Previous studies tentatively attributed this phenomenon to either strengthened backbone H-bonds or to changes in protein-solvent interactions. To help unravel these contributions, we performed Collision Induced Unfolding experiments (CIU) on gaseous proteins. The indistinguishable CIU profiles of deuterated and unlabeled proteins suggest that D2O-induced stabilization originates from solvent effects.