Development of HDX-MS methods to further investigate complex protein samples

Thesis

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) has become an essential technique in biological research for investigating the dynamic structural changes in proteins in response to various perturbations. This thesis explores the structural alterations that occur when proteins interact with different natural binding partners, aiming to identify and understand these differences. A significant contribution of this work is the development of a novel analytical script that streamlines the HDX-MS process. By integrating statistical testing, this tool rapidly identifies significant changes and presents the results clearly and concisely, enhanced by visual aids that facilitate the interpretation of structural variations across different experimental states. The thesis also includes several case studies of protein complexes analysed using HDX-MS, which had not previously been investigated with this technique. These findings were corroborated with complementary methods and aligned with expectations based on prior research.

Additionally, this thesis addresses the challenges posed by glycosylation, a post-translational modification that often complicates data interpretation and analysis, leading to gaps in sequence coverage. To overcome this, a method is presented that effectively manages glycan moieties, significantly improving the accuracy and completeness of glycoprotein analysis. This approach includes the use of a glycosidase that is active under typical quench conditions, this enables glycan cleavage while preserving the integrity of the native data throughout the labelling process. As a result, sequence coverage is enhanced, allowing for a more comprehensive understanding of the entire protein structure. Overall, this work offers advancements in both the analytical and practical aspects of HDX-MS, contributing to a deeper understanding of protein dynamics and the impact of glycosylation on protein behaviour.

Authors: Smith, L

• DOI: 10.5287/ora-5zzx61j1e
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: HDX-MS; SHIP2; mass spectrometry; analysis; SHIP1; structural biology; IPNS
• Subjects: Biology; Chemistry