Protein footprinting is a technique used to determine changes in higher order structure in proteins. Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), Fast photochemical oxidation of proteins (FPOP) and synchrotron footprinting are common footprinting methods utilising different mechanisms to achieve elucidation of higher order structure. This thesis aims to probe the functionality of these methods, and to improve understanding of the mechanisms of each approach. HDX-MS is used to probe the action of polysorbates on antibodies, the effect of local residues is probed in FPOP, and the viability of synchrotron footprinting is probed for binding of TiO2 to MtrC.
Polysorbates are commonly used excipients in pharmaceutical formulations, utilised to increase stability of active pharmaceutical ingredients (APIs). Despite this, the molecular mechanism of their stabilizing effect is not well described in literature. When used above their critical micelle concentration (CMC), they can protect against aggregation, in particular shielding hydrophobic patches of proteins. In protein formulations, polysorbates 20 and 80 are typically above the polysorbate CMC.
Here, using a common and abundant model protein, myoglobin, as well as a set of monoclonal antibodies with varying tendencies to aggregate, the principle of this protective effect in molecular detail is examined. Myoglobin was selected as a model protein as it is well characterised and not aggregation prone. Experiments are carried out with polysorbate excipient added both above and below the CMC, and long term (storage) stability and the extent of possible oxidative damage are also investigated.
Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a structural mass spectrometry technique that utilises the exchange of solvent accessible backbone hydrogens to determine changes in protein structure, through differences in the uptake of deuterium based on solvent accessibility. These can be used to infer structural changes within the protein between states. Further, as access to solvent accessibility is affected by protein binding, HDX-MS can be utilised to map binding sites for both protein-protein interactions and binding with synthetic molecules.
By utilising HDX-MS, structural changes on proteins due to polysorbate exposure can be determined, and regions of protection and deprotection identified.
Initially, myoglobin samples with and without polysorbates were analysed without incubation by HDX to determine the veracity of the method for polysorbate analysis. This analysis showed no difference in protein structure with the addition of fresh polysorbate 20 and 80. This suggests polysorbate does not immediately perturb structure on addition to myoglobin both locally and globally, which is anticipated with a stable protein such as myoglobin.
Investigations of the antibodies WFL and STT, with WFL being more aggregation prone than STT, showed polysorbate 80 perturbs the structure of WFL in comparison to polysorbate 20 and the control. This is likely due to polysorbate 80 having high affinity to hydrophobic pockets on the fc domain of WFL.
FPOP is another structural mass spectrometry technique that allows for determination of conformational changes, similar to HDX. However, the effect of the local environment on labelling has not been rigorously scrutinised. Here, I look at six peptides, with varying distance between tryptophan and phenylalanine residues, and determined the total oxidation of each peptide.
Here, we see for the first time evidence to suggest fundamentally that as two highly oxidisable residues are spatially located closer, total oxidation decreases. It is hypothesised this is due to competition between two reactive residues for a limited number of hydroxyl radicals. As the distance increased between the residues, it is proposed the local competition between residues for the limited number of hydroxyl radicals is decreased due to spatial distance, and thus more labelling can occur at each reactive residue.
Finally, MtrC binding with TiO2 is interrogated through synchrotron footprinting. Here for the first time I determine that the location of binding of TiO2 to MtrC is primarily due to electrostatic interactions, as opposed to chemical binding.
This provides a solution for rational design of light harvesting particles, and how to determine binding of these molecules for future molecular battery design.
Thus, each footprinting method is interrogated and understanding of each method has been advanced within this thesis.
