New technologies for cross-linking mass spectrometry

Thesis (Ph.D.)--University of Washington, 2022Cross-linking mass spectrometry is a rapidly evolving technique for obtaining structural information about proteins and protein complexes in their near native state in a high-throughput manner. Cross-linked peptides are challenging to identify due to being low abundance analytes that produce complex fragmentation spectra. While cross-links can provide valuable structural data, these challenges mean that current technologies can sample only a small portion of the protein structures and assemblies that exist in complex systems.In this work I demonstrate three new technologies I developed to enhance cross-linking mass spectrometry experiments. The first section describes the development of the cross-linking search tool, Mango, which enables identification of cross-links in complex samples generated from a variety of cross-linkers. The next section discusses the development of a tetrameric cross-linker and its application in studying the mitochondrial interactome from murine hearts. Higher dimensional cross-linkers move experiments from binary interactions to ternary and quaternary interactions, helping to better characterize interfaces composed of many proteins. The final section describes the development of software to facilitate liquid chromatography coupled to tandem mass spectrometry experiments in a Fourier transform ion cyclotron resonance array mass spectrometer by automating ion selection, ion transfer, and analog signal processing for each cell which allows for parallel acquisition of high-resolution mass spectra. While this hardware has been previously described, modifications to the instrument’s ion handling and analog signal processing enable cross-linking experiments to be carried out with parallel detection and an enhanced duty cycle