Development and Application of Novel Software and Methods to Study Carbohydrates, Proteins and Protein Complexes Using Mass Spectrometry and Ion Mobility Mass Spectrometry Methods

Abstract

Gas phase techniques, such as Mass Spectrometry (MS) and Ion Mobility-Mass Spectrometry (IM-MS) have proven themselves to be exceptionally useful tools in the analysis of a range of biomolecules such as glycans, proteins or protein complexes. The popularity of MS-based methods can be attributed to low-sample consumptions, high-sensitivity and high-throughput and with the inclusion of Ion Mobility (IM) separation, the composition of complex samples can be determined with increased certainty. Further to this, IM provides additional details about the analytes, in particular revealing information on their size and shape. IM-MS measurements yield the typical mass-to-charge (m/z) information and the rotationally averaged Collision Cross Section (CCS) that is a descriptor of the analytes conformation. CCS values not only reveal information about the size and shape of the molecule but also about the "global" conformational dynamics of the measured ion, hence it can also be described as a CCS distribution (CCSD). The results of IM-MS measurements can be readily interfaced with molecular modelling, where experimentally derived CCSs are compared to computationally derived values from an ensemble of structures. In the first instance, we investigated the impact of partial atomic charges on the computed CCSs on a set of small carbohydrate molecules and determined that selection of the computational model can have a significant impact on the computed CCSs, potentially affecting structural assignment. We then investigated how the position of charged residues in the primary sequence of three intrinsically disordered proteins (IDPs) affected their conformational diversity when measured using MS and IM-MS. Following this, we investigated gas phase unfolding (also known as collision induced unfolding) to uncover the stabilizing effects of a cofactor, prFMN, on the dimeric protein Fdc1UbiX. The popularity of activated Ion Mobility (aIM), either in the form of collisional activation (CIU) or surface activation (SID) led us to develop novel methodologies that can be deployed on commercial instruments such as Waters Synapt G2/G2S/G2Si, automating and simplifying the acquisition process. The introduction of new methods also resulted in the development of new software tools that are capable of both the analysis and interpretation of data, facilitating the development of a software package ORIGAMI. This software has grown in size and complexity, now enabling interrogation of a broad range of MS and IM-MS datasets as well as becoming a powerful visualisation package that is capable of producing interactive documents that can be a stepping stone towards more openness and transparency within the MS and IM-MS community.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Munro (Supervisor) & Perdita Barran (Supervisor)