Pre-stabilization strategies for systems-wide in situ crosslinking mass spectrometry

Michael, Andrew Roy

Pre-stabilization strategies for systems-wide in situ crosslinking mass spectrometry

dc.contributor.advisor	Schriemer, David C.
dc.contributor.author	Michael, Andrew Roy
dc.contributor.committeemember	Lees-Miller, Susan P.
dc.contributor.committeemember	Williams, Gareth J.
dc.date	2025-02
dc.date.accessioned	2025-01-14T16:10:43Z
dc.date.available	2025-01-14T16:10:43Z
dc.date.issued	2025-01-09
dc.description.abstract	Understanding the associations proteins form within a network of protein-protein interactions (PPIs) provides insight into the functions that drive cellular processes. These networks have largely been sampled and studied using various high-throughput screening techniques providing network maps. However, these investigative approaches only generate indirect interaction data that are prone to false positives. Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome with high resolution and coverage. Current in situ XL-MS methods are limited by the low permeability of crosslinking reagents necessitating long incubation times to accrue appreciable signal. Consequently, sampling of the interactome is sparse and the long incubation times can distort the structural proteome resulting in questionable validity of the identified PPIs. This thesis aims to address these concerns through development of novel in situ XL-MS procedures that incorporate pre-stabilization into the workflow to facilitate cellular ultrastructure preservation and improve sampling of the spatiotemporal proteome. Pre-stabilization of cells was explored through chemical and temperature-based fixation strategies using either formaldehyde or freeze-substitution, respectively. Both methods facilitated PPI sampling at or above conventional in situ XL-MS workflows and provided opportunities to further maximize crosslinking yields while maintaining a preserved cellular ultrastructure. Pre-stabilizing cells with formaldehyde further created an opportunity to explore a novel crosslinking approach where the crosslinking reaction was split into two sequential and orthogonal coupling events. This approach followed a stepwise process of installing crosslink precursors across the proteome at saturated levels with crosslink formation done through click-chemistry, resulting in over 5X higher yield of PPIs over conventional in situ XL-MS.
dc.identifier.citation	Michael, A. (2025). Pre-stabilization strategies for systems-wide in situ crosslinking mass spectrometry (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.
dc.identifier.uri	https://hdl.handle.net/1880/120420
dc.language.iso	en
dc.publisher.faculty	Graduate Studies
dc.publisher.institution	University of Calgary
dc.rights	University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subject	Mass Spectrometry
dc.subject	Crosslinking Mass Spectrometry
dc.subject.classification	Biology--Molecular
dc.subject.classification	Biochemistry
dc.title	Pre-stabilization strategies for systems-wide in situ crosslinking mass spectrometry
dc.type	doctoral thesis
thesis.degree.discipline	Medicine – Biochemistry and Molecular Biology
thesis.degree.grantor	University of Calgary
thesis.degree.name	Doctor of Philosophy (PhD)
ucalgary.thesis.accesssetbystudent	I do not require a thesis withhold – my thesis will have open access and can be viewed and downloaded publicly as soon as possible.