Schriemer, David C.Raval, Shaunak2023-09-262023-09-262023-09-13Raval, S. (2023). Enabling structural proteomics with high efficiency protein enrichment technology (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.https://hdl.handle.net/1880/117130https://doi.org/10.11575/PRISM/41972The functional state of proteins is inherently flexible, which allows them to interact with other biomolecules, including other proteins, to carry out many of their cellular functions. Understanding the structural dynamics of proteins and their network of associations is key to understanding their role in biology. Proteomics, the collection of mass spectrometry (MS)-based techniques to study proteins, provides a broad view of the organization of protein structure, from an individual dynamic unit to large-scale multiprotein assemblies, enabled by the application of labelling chemistries. This dissertation presents novel analytical workflows and data analysis routines to overcome current challenges in proteomics methods for the identification of protein-protein interactions (PPIs) and the study of protein conformation and dynamics. Affinity purification followed by mass spectrometry (AP-MS) is a prominent approach in the study of PPIs. However, the conventional workflow suffers from low enrichment efficiencies. I present and evaluate a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing non-specific binding. It enables the study of protein conformational analysis directly from cells as I demonstrate first by describing new concepts in data analysis for hydrogen/deuterium exchange mass spectrometry (HX-MS) and second by applying them to proteins isolated directly from cells.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.Affinity PurificationMass SpectrometryHydrogen-Deuterium ExchangeBioanalytical ChemistryChemistry--AnalyticalBiochemistrydoctoral thesis