Fibrillization and Crystallization of Small Molecule Assembly Probed by Ion Mobility Spectrometry-Mass Spectrometry and Complementary Techniques

Despite the advances in understanding the amyloid cascade hypothesis and its extension to other small molecules, the mechanism underlying the initiation and progression of rare metabolic disorders is yet to be fully understood. Current knowledge highlights amyloid fibrils as the primary cytotoxic species in the cascade of events of the disease, with little known about the early intermediate oligomeric species. This dissertation primarily focused on exploring early oligomeric species overlooked by the amyloid cascade hypothesis by employing ion mobility spectrometry-mass spectrometry to understand their structure and conformation.

My initial PhD years focused on method development before the biophysical studies. Thus, in the first part of this dissertation, we probe the adduction of Zn²⁺ and Ca²⁺ to mixed m-xylene urea/thiourea macrocycle. Our results showed unique intermediate complexes with ZnCl₂ while binding to Ca²⁺ stabilized large oligomers of the macrocycle, a phenomenon absent with Zn²⁺. Additionally, in N-pyridyl bis-urea macrocycles, the position and proximity of the pyridyl nitrogen to the urea-carbonyl were critical factors to the formation of charge isomers (protomers). Thus, showing the capability of IMS-MS as a robust and inexpensive analytical toolbox for elucidation of structure-behavior and interaction in gas phase.

The second part of this dissertation focuses on the aggregation mechanisms of small metabolites and the growth process of their supramolecular assemblies in gas phase. With the paradigm shift revealing amyloid formation by non-disease related metabolite in inborn error of metabolism (IEM), different small metabolites were studied i.e., homocysteine, glycine and uric acid to investigate their self-assembly and gas-phase behavior and how their self-assembled species transform to cytotoxic species involved in the disease etiology. We take advantage of other complementary techniques in addition to IMS-MS to further explore these species in the disease progression.

Lastly, IMS-MS was employed to elucidate the crystallization process providing insights into the plausible underlying mechanism critical for nucleation and growth of crystals. Using adenine as our model of choice, the cluster necessary to form the critical nucleus was identified i.e., specific oligomeric species crucial for the nucleation of the crystal. In conclusion, we have successfully implemented ion mobility spectrometry-mass spectrometry to elucidate complex systems.