Protein assemblies represent the workhorses of the cell, forming the basis of all cellular processes. Their biological roles are intimately associated with their topologies, making the structural elucidation of proteins and protein complexes a critical requirement to understanding their function. While traditional structural biology approaches have greatly contributed to our current understanding of protein structure, they are ill-suited for analyzing the conformational dynamics associated with heterogenous protein complexes and their protein-protein interactions (PPIs). As a result, there is a growing demand for the development of new structural approaches to elucidate the impact of protein dynamics on the regulation of integral biological processes required for cell homeostasis. In particular, cross-linking mass spectrometry (XL-MS) has arisen in recent years as a popular hybrid structural technique for the topological determination of conformationally and compositionally heterogenous protein complexes. However, most studies utilizing cross-linking thus far have been limited to the determination of static protein structures.

Here, I focus on the development and application of quantitative cross-linking mass spectrometry (QXL-MS) strategies to determine how conformational dynamics of cullin-RING ligases (CRLs) dictate and regulate their ubiquitinating activity. Proteasomal dysregulation has been associated with a wide range of human pathologies, from diabetes and various forms of cancer to autoimmune and neurodegenerative disorders. As a result, the PPIs associated with CRL assemblies represent potential targets for therapeutic intervention. A comprehensive understanding of E3 ligase structure and conformational dynamics is critical for the development of pharmacological drugs that selectively inhibit or upregulate their function. However, such heterogenous assemblies are notoriously difficult to study using traditional structural biology techniques.

While this thesis focuses on the application of quantitative XL-MS strategies to study cullin-RING ligase machinery, these platforms represent versatile methodologies that can be universally employed for structural studies on a wide range of protein systems.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a series novel sulfoxide-containing cross-linker that target acidic residues (dihydrazide sulfoxide (DHSO)), cystine residues (bismaleimidesulfoxide (BMSO)), and finally a heterobifunctional cross-linker that targets lysine on one end and a non-specifically targets residues (Succinimidyl diazirine sulfoxide (SDASO)) on the other. We demonstrate that cross-linkers create cross-linked peptides that display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Furthermore, we applied these linkers to either characterize the yeast 26S proteasome (SDASO) or the Cop9 signalosome (DSSO, DHSO and BMSO) demonstrating both the feasibility of SDASO’s photocross-linking of large protein complexes for the first time and the ability of multi-chemistry data for the integrative structure modeling of protein complexes to determine the interaction and structural dynamics of CSN, respectively. Moreover, our platform targeting various chemistries with cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of multi-chemistry targeting approach to studying protein complexes.

Cross-linking mass spectrometry maps the structural topology of protein complexes by using

the distance between linked residues as spatial constraints, complementing other structural

biology techniques. However, the identification of cross-linked peptides scales poorly with the

number of proteins analyzed. Our lab has previously developed MS-cleavable cross-linkers to

enable the separation of cross-linked peptides prior to sequencing, enabling peptide identifica-

tion using standard peptide search databases. We describe the design and implementation of

platform and application named XLTools for the automated identification of MS-cleavable

cross-linked peptides. XLTools supports open and proprietary data formats and common

peptide search databases, facilitating its integration into existing workflows. Furthermore,

we developed peak-picking and validation algorithms to enable the accurate quantitation of

cross-linked peptides in complex samples. We demonstrate the application of XLTools to

the quantitative analysis of the 26S proteasome cross-linked in vivo and in vitro.