Development and Application of HDX-MS Methods in Biopharmaceuticals

In this Thesis work, I investigated the intricate relationship between protein structure, dynamics, and function, emphasizing the need for advanced techniques to study these aspects. Traditional methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy face limitations, leading to the development of alternative tools such as mass spectrometry (MS) with hydrogen-deuterium exchange (HDX). This PhD project focuses on applying HDX-MS to investigate protein dynamics in the field of coagulation and amyloidosis. Chapter 3 delves into the exploration of haemadin, a natural protein anticoagulant, using HDX-MS and other techniques. The research aims at better characterizing its interaction with thrombin, in order to develop a novel thrombin inhibitor, with significant findings on haemadin's structure, function, and chemical synthesis of a novel thrombin inhibitor. Chapter 4 examines thrombin mutations and their role in anti-thrombin resistance, providing insights into coagulation disorders. HDX-MS studies reveal increased flexibility in critical thrombin residues, contributing to the understanding of molecular mechanisms underlying anti-thrombin resistance induced by specific mutations. The final chapter, Chapter 5, focuses on transthyretin amyloidosis, a group of diseases characterized by the accumulation of misfolded proteins. The study investigates the impact of amino acid mutations on transthyretin structure, stability, and susceptibility to proteolysis using various analytical techniques, including HDX-MS. The results suggest a correlation between structural alterations, unfolding stability, and proteolysis susceptibility, shedding light on the pathogenic mechanisms leading to fibril formation. In summary, the research employs advanced techniques such as HDX-MS to comprehensively explore protein dynamics in coagulation, anti-thrombin resistance, and transthyretin amyloidosis, contributing valuable insights to the understanding of these complex biological processes.