Master of Science
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.
Summary for Lay Audience
Proteins are molecules that perform various biological roles important to all living organisms. The function of these molecules depends on their overall structure. Many techniques are available to probe protein structure and conformational dynamics. These include native ESI as well as chemical labelling, in combination with MS. Unfortunately, many fundamental aspects of these techniques remain poorly understood. For every ESI experiment, proteins have to be converted to gaseous ions. This process is carried out by applying high voltage to an ESI capillary where a protein solution is introduced, generating a plume of highly charged droplets that ultimately release protein ions into the gas phase. The extent to which gaseous proteins and non-covalent complexes maintain their native structures depends on how gentle the ESI conditions are. While Native ESI is a useful tool for probing protein structure, it does not provide high-resolution data. Chemical labelling in conjunction with MS fills this role by probing solvent accessible surfaces. The question to what extent these labels are benign has been the subject of debate for decades.
In this work, we investigate ESI parameters that can affect the outcome of protein experiments. The results of chapter 2 demonstrate that cone voltage, and solvent conditions can greatly influence whether proteins retain native-like structures. Moreover, we discussed the chemistry of NH4Ac (a widely used ESI additive). This chemical has a little buffering capacity at neutral conditions but can stabilize the pH at slightly acidic values. Our data reveal that globular proteins like cytochrome c and lysozyme can tolerate such slight pH drops.
In chapter 3, we shed light on HDX (a widely used chemical labelling method) and scrutinize the assumption that labeling proteins with deuterium is benign. We investigated the purported stability of deuterated proteins in the solution phase using thermal unfolding studies. Similarly, we utilized collision-induced unfolding to investigate the stability of deuterated proteins in the gas phase. Our data demonstrates that exposure to D2O might alter certain aspects of protein structure and dynamics in solution. In contrast, gas-phase experiments revealed that the stability of gaseous protein ions is indistinguishable before and after deuteration.
Haidar, Yousef, "Protein Stability in Solution and in the Gas Phase." (2023). Electronic Thesis and Dissertation Repository. 9673.