Schriemer, David C.Michael, Andrew Roy2025-01-142025-01-142025-01-09Michael, 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.https://hdl.handle.net/1880/120420Understanding 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.enUniversity 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.Mass SpectrometryCrosslinking Mass SpectrometryBiology--MolecularBiochemistrydoctoral thesis