Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.772036
Title: XLIM-MS : towards the development of a novel approach to cross-linking mass spectrometry
Author: James, Juliette M. B.
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Abstract:
In the cellular environment proteins form diverse and complex networks of interactions that control a variety of vital functions. Understanding the structure of these multi-subunit assemblies is key to understanding their function. To date the majority of structural information has been obtained through crystallographic studies, electron microscopy and NMR. However, the size and dynamics of these macro-molecular machines often precludes their analysis by such traditional methods. Cross-linking mass spectrometry offers a complementary structural technique. The distance restraints provided by the technique are formed in solution and offer more native structural information. Mapping of these restraints allows determination of the relative positions of amino acid residues in wider three dimensional structures. To date the analysis of cross-linked peptides has almost exclusively been conducted with Orbitrap analysers. As a result most of the software applications designed to identified cross- link:spectrum matches have been developed with data from this type of analyser. Here we present an optimised protocol for the analysis of cross-linked samples using a Quadrupole Time-of-Flight mass spectrometer (QToF). We show that existing software can be configured to analyse QToF data with minimal adaptations. We evaluate the usefulness of the xQuest linear discriminant score in determining genuine cross-link:spectrum assignments. The increased size and charge of crosslinked peptides compared to their un-crosslinked counterparts makes them ideal candidates for separation by ion mobility mass spectrometry. We take advantage of the unique geometry of the Triwave Stacked Ring Ion Guide to explore the effects of ion mobility separation on both precursors and fragment ions from cross-linked samples. To evaluate the sequence coverage and signal to noise ratio of identified cross-link:spectrum matches we present two computational solutions: ValidateXL and AnnotateXL.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.772036  DOI: Not available
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