Ultimately, the work outlined within this dissertation demonstrates the development of SID, IM, and UVPD instrumentation and methods, expanding the tools available within native MS to perform more in-depth characterization of peptide, protein, and protein complex structures.

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

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 utility of 193 nm ultraviolet photodissociation (UVPD) is evaluated for high-throughput proteomics applications including: analysis of small peptides in a traditional bottom-up proteomics workflow, analysis of heavily modified larger middle down sized peptides, and heavily modified intact proteins in a top-down proteomics workflow. UVPD uses higher energy ultraviolet photons (193 nm, 6.4 eV per photon), which are absorbed by the backbone to activate and dissociate ions effectively. UVPD dissociation is able to generate extensive backbone fragmentation enabling excellent characterization of peptides and proteins compared to traditional methods. Moreover, UVPD is also less hindered by certain experimental variables such as degree of modification, charge state and even ion polarity. These features are easily capitalized on for proteomics applications especially analysis of post translational modifications (PTM's). Characterization of PTM's is of great interest due to their involvement in several important cellular processes including cell signaling, tumorigenesis and gene expression. The studies covered in this work focus on utilizing the unique capabilities of UVPD to: 1.) characterize underrepresented peptides (acidic peptides and phosphopeptides) in the negative polarity including development of software for the analysis of the data generated, 2.) analyze intact proteins which have undergone extensive chemical modification and charge state augmentation, and 3.) precisely characterize histone proteins which are heavily modified due to their central role in gene expression and other transcription related functions.

Characterization of macromolecular protein assemblies by collision-induced and surface-induced dissociation: Expanding the role of mass spectrometry in structural biology.