James, Juliette M.B.;
(2019)
XLIM-MS Towards the Development of a Novel approach to Cross-linking Mass Spectrometry.
Doctoral thesis (Ph.D), UCL (University College London).
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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.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | XLIM-MS Towards the Development of a Novel approach to Cross-linking Mass Spectrometry |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10068144 |
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