Structural biology studies aimed at the elucidation of protein-dependent disease mechanisms have traditionally relied on high-resolution techniques, including X-ray crystallography, nuclear magnetic resonance, and cryogenic electron microscopy. While such methodologies remain standard for gaining information on the core structure of proteins, specific drawbacks including time or large sample quantities associated with these approaches have spawned the development of other pipelines. Mass spectrometry (MS) is one such tool that has gained traction as a rapid and sensitive low-resolution structural biology technique. Routinely protein complexes of interest are reacted in solution with covalent chemical probes to preserve structural information prior to enzymatic digestion and mass spectrometric read-out. However, with the advent of native MS, protein complexes can now be efficiently transferred intact into the gas phase using high ionic strength solutions while retaining structures reminiscent of their solution conformations, and directly interrogated using MS/MS methods. Ultraviolet photodissociation (UVPD) is one such ion activation method that has been extensively developed to break apart protein complexes in a manner that allows conclusions about structure to be drawn based on the fragmentation behavior. The work presented here leverages native mass spectrometry in conjunction with 193 nm UVPD to probe a variety of biologically important protein-ligand and protein-protein complexes. The utility in a native UVPD-MS approach for structural examination of protein-ligand complexes is demonstrated through characterization of conformational changes associated with the catalytic cycle of a phosphotransferase enzyme as well as elucidation of structural changes resulting from mutation or inhibition of an enzyme responsible for conferring antibiotic resistance to bacteria. An oncogenic protein and several clinical variants bound to a downstream effector protein provides an example of the capabilities of native MS and UVPD to characterize the structure of a protein-protein complex. Native UVPD-MS is also used for epitope mapping of the main antigenic determinant of the influenza virus. Aimed at improving analysis of larger complexes, multistage native UVPD-MS is developed to probe the structure of a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Lastly, uniting on-line capillary electrophoresis (CE) with multistage native UVPD-MS offers a high-throughput workflow for structural characterization of ribosomal protein complexes

The work detailed in this dissertation describes the advantages that 193 nm ultraviolet photodissociation (UVPD) affords for characterization of structurally complex biological molecules as compared to traditional ion activation techniques, such as collisional or electron-based dissociation, for mass spectrometry. UVPD, either alone or in tandem with collisional activation such as collision induced dissociation (CID), consistently provides more extensive structural information about biomolecules. One such system where the utility of both UVPD and CID was employed was in the structural characterization of lipid A species. Lipid A, the innermost structural component of lipopolysaccharides (LPS) which decorate the surface of Gram-negative bacteria, may undergo covalent modifications in order to provide resistance to antibiotics. By utilizing a combinatorial approach, CID is able to characterize the covalent modifications that are present while UVPD is able to elucidate which side of the molecule (reducing or nonreducing end) undergoes the modification through selective fragmentation of the diglucosamine backbone. This approach confirmed the presence of aminoarabinose modification present on the LPS of A. baumannii after exposure to the antibiotic polymyxin B. Another instance of utilizing the power of both photodissociation and collisional activation was in the characterization of oligosaccharide molecules from LPS of E. coli. These biomolecules are typically heavily phosphorylated near the reducing end of the saccharide backbone, and as such, collisional activation produces fragment ions originated from cleavages localized near the phosphate sites. UVPD of the oligosaccharides produces a plethora of diagnostic fragment ions throughout the molecule, but this often leads to spectral congestion and ambiguous fragment assignment. UVPD generates charge-reduced precursor ions that can be subjected to subsequent collisional activation in a MS3 event, allowing complete characterization significantly fewer confounding product ions as compared to UVPD alone. Another hallmark of UVPD is its fast, high energy deposition that causes cleavage of covalent bonds while allowing survival of non-covalent interactions. This characteristic allows electrostatic interactions to be mapped in non-covalent complexes, unlike the collisional activation which preferentially cleaves weak non-covalent interactions owing to the stepwise nature of collisional activation. In this work, it is demonstrated that UVPD of the electrostatic complex between a cationic antimicrobial peptides (CAMP) and Kdo2-lipid A illuminates, through the production of diagnostic holo peptide fragment ions retaining the intact mass of the lipid A species, which amino acids in the peptide sequence are responsible for mediating the interaction between the two molecules in the gas phase. In contrast, collisional activation of the electrostatic complex between the two species simply results in the disruption of the network of non-covalent interactions, only yielding apo peptide product ions. In the same vein, this notion of retention of electrostatic interactions post-photodissociation was employed to interrogate where metal ions were sequestered in proteins. UVPD has previously been touted as a means to determine the binding location of ligands (such as drug molecules) to proteins after transporting the protein-ligand complexes to the gas-phase by native ESI. This methodology was extended to determine the binding location of metal ions (such as calcium, copper, silver, and praseodymium, to name a few) to proteins. The binding sites of calcium (II) and a series of lanthanide (III) ions were successfully determined for staphylococcal nuclease, the binding sites of silver (I) and copper (II) were determined for azurin, and multiple binding sites for calcium (II) and select lanthanides (III) were determined for calmodulin, all agreeing with reported crystal structure data. These are but only a few examples of the utility of UVPD as an alternative to ion activation in the gas phase. The unprecedented characterization of ions by UVPD, regardless of polarity, number of charges, size of the molecule, or molecular interactions present, suggests that there are many other potential applications of UVPD in the future

The definitive guide to mass spectrometry techniques in biology and biophysics The use of mass spectrometry (MS) to study the architecture and dynamics of proteins is increasingly common within the biophysical community, and Mass Spectrometry in Structural Biology and Biophysics: Architecture, Dynamics, and Interaction of Biomolecules, Second Edition provides readers with detailed, systematic coverage of the current state of the art. Offering an unrivalled overview of modern MS-based armamentarium that can be used to solve the most challenging problems in biophysics, structural biology, and biopharmaceuticals, the book is a practical guide to understanding the role of MS techniques in biophysical research. Designed to meet the needs of both academic and industrial researchers, it makes mass spectrometry accessible to professionals in a range of fields, including biopharmaceuticals. This new edition has been significantly expanded and updated to include the most recent experimental methodologies and techniques, MS applications in biophysics and structural biology, methods for studying higher order structure and dynamics of proteins, an examination of other biopolymers and synthetic polymers, such as nucleic acids and oligosaccharides, and much more. Featuring high-quality illustrations that illuminate the concepts described in the text, as well as extensive references that enable the reader to pursue further study, Mass Spectrometry in Structural Biology and Biophysics is an indispensable resource for researchers and graduate students working in biophysics, structural biology, protein chemistry, and related fields.

Ultraviolet photodissociation (UVPD) is an alternative high-energy ion activation technique implemented to produce information rich tandem mass spectra. Dissociation of biomolecules by UVPD results in structure dependent fragmentation to reveal molecular details that are otherwise undiscernible by traditional tandem mass spectrometry techniques, providing an avenue to rapidly interrogate the structure-function relationship of biologically relevant species. Applied to glycerophospholipids, UVPD is capable of resolving locations of unsaturation and stereospecific numbering of acyl chains, subtle structural features that are traditionally challenging to resolve. In the analysis of intact proteins, UVPD produces excellent sequence coverage that can pinpoint sites of post translational modifications, while providing conformation sensitive fragmentation that also informs changes in higher-order structure that occur upon ligand binding or mutations. Studies covered in this work extend the unique capabilities of UVPD to characterize increasingly complex molecules, explore associations between UVPD resolved structure and disease, and develop an understanding of dissociation mechanisms that govern fragmentation induced by 193 nm photons. Here, the high versatility of this technique was applied to the detailed structural characterization of cardiolipins at the double bond and stereochemistry level by utilizing hybrid techniques that combine collisional activation with UVPD; similarly, UVPD was integrated to both imaging and chromatographic workflows to evaluate fatty acid structure and phosphatidylcholine structure, respectively, as a function of disease state; furthermore, fragmentation of intact proteins was evaluated to discern mechanisms that influence photon-induced dissociation and leveraged to assign paratopes and interpret complex top-down spectra of proteins with disulfide bonds