The development of structural mass spectrometry based techniques for the study of aggregation-prone proteins.

The structural characterisation of aggregation-prone proteins, and the development of methods which allow such investigations, is of paramount importance to the prevention and treatment of illnesses such as Alzheimer’s disease, and to the development of the next generation of biotherapeutic medicines. This thesis outlines the development of several structural mass spectrometry based techniques, including hydrogen deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP) to study the structure and dynamics of two protein systems: β2 – microglobulin (β2m), along with two of its variants (ΔN6 and D76N), as well as WFL and STT, two biotherapeutic antibody variants. The first aggregation-prone variant of β2m studied, missing the six N-terminal residues (ΔN6), showed significant structural changes compared with the wild-type protein close to the truncation site when examined by FPOP-LC-MS/MS, consistent with experiments performed using more well-established structural MS methods, such as HDX. A thorough examination of the data revealed the presence of positional isomers, generated from the oxidation of aromatic amino acids within peptides, which showed oxidation patterns consistent with known structural changes observed by NMR, indicating sub-amino acid level resolution may be achievable using FPOP. The second aggregation-prone variant, D76N, showed significant structural changes proximal to the site of the amino acid substitution when using HDX, but only minimal changes when analysed by FPOP-LC-MS/MS. We hypothesise that these changes may be due to a disruption of hydrogen bonding networks within the loop containing the amino acid substitution, and that these differences may lead, or contribute, to the increased aggregation propensity of the D76N variant, relative to the wild-type protein. FPOP-LC-MS/MS analysis of the antibody variants WFL and STT, so called due to their different amino acid sequences in the heavy chain complementarity determining regions (CDRs), revealed long-range conformational changes at the interface between the two constant domains of the Fab arm, a considerable distance from the site of the amino acid substitutions. Although the relationship between these observed changes and the propensity of WFL to undergo reversible self-association cannot be determined from these data, these experiments strongly indicate that conformational changes can be transmitted between the antigen binding region and the constant domains of the Fab arm – two regions often considered to be functionally independent. The large data set obtained from these experiments allowed further development of the FPOP experimental technique, where trends in the altered hydrophobicity of modified peptides, probed by relative retention time shifts of peptides following separation by reverse-phase chromatography, highlight the possibility of using such analyses to aid in the assignment of modified species, when coupled to tandem MS analysis. Overall, the data presented in this thesis shed new light on the changes in protein structure and dynamics associated with the two aggregation-prone variants of β2m, as well as the aggregation-prone mAb, WFL which may provide key insights into the causes of aggregation or reversible self-association in these proteins. Similarly, the work presented here contributes significantly to a greater understanding of FPOP, the application of both FPOP and HDX towards the study of aggregation-prone proteins, and provides a foundation for the further advancement of these methods in future research