Structural characterization of complex biological systems via ultraviolet photodissociation mass spectrometry

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 futureChemistr