Photon-based tandem mass spectrometry provides a versatile ion activation strategy for the analysis of polypeptides, proteins, and lipids. 351-nm ultraviolet photodissociation mass spectrometry (UVPD-MS) is a facile and selective tandem dissociation technique used to elucidate chromophore-modified peptides within large mixtures. A bis-aryl chromogenic chemical probe was utilized to target solvent exposed primary amine residues within native protein states. Collision-induced dissociation (CID) was employed to indiscriminatly characterize the complete proteolytic digest while chromophore containing peptides were selectively dissociated with 351-nm UVPD; thus streamlining the identification of targeted peptides with structurally informative residues. Protein amine residue reactivities were then compared with predicted solvent exposures to elucidate protein tertiary structures, their mechanistic properties, and ligand-binding interactions. High-energy 193-nm UVPD is a fast, high-energy tandem mass spectrometry method and frequently generates fragment ions typically inaccessible to CID-based methods. Native mass spectrometry was coupled to top-down 193-nm UVPD for the gas phase characterization of non-covalent protein-ligand and protein-protein complexes. This method yielded a unique array of fragment ions for a comprehensive analysis of protein structures. UVPD of non-covalent complexes generated many polypeptide backbone fragments to characterize the primary sequence of proteins. Furthermore, top-down UVPD engendered cleavages with intact electrostatic interactions; this provided insight into the binding interfaces within protein-ligand complexes and the higher order structural architectures of oligomeric complexes. High-resolution 193-nm UVPD was paired with high performance liquid chromatography (LC) for the streamlined structural analysis of amphiphilic glycolipids within complex mixtures. For all glycolipids, UVPD provided the most comprehensive structural analysis tool by affording a diverse array of fragment ions to characterize both hydrophobic and hydrophilic moieties. UVPD based LC-MS separations of gangliosides shed light on the ceramide lipid bases, glycan moieties, and their isobaric structural variants. UVPD activation of lipid A and lipooligosaccharides (LOS) compounds generated a mixture of C-C, C-O, and C-N fragment ions to illustrate the hydrophobic acyl structures, while cleavages within the glycosidic, and cross-ring cleavages allowed the determination of acylation patterns. Novel LC-MS separation strategies were developed to elucidate and structurally characterize complex mixtures of lipopolysaccharide containing compounds.

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 dissertation will discuss the advancement of informative structural biology techniques utilizing a top-down centric workflow with 193nm ultraviolet photodissociation (UVPD) mass spectrometry. Native electrospray ionization is used to transport proteins to the gas phase in a native-like state, then UVPD is used for structural characterization to reveal ligand binding sites within a protein-ligand complex as well as detect conformational changes based upon the suppression or enhancement of backbone cleavages. Conformational changes induced by ligand exchange or removal and single amino acid mutations as well as combinations of the two (ligands and mutations) are investigated. The rich fragmentation patterns of UVPD are also used for structural characterization of crosslinked proteins. Typically these crosslinking experiments are performed by bottom-up mass spectrometry with has significant shortcomings. The main drawback is the need for proteolysis which cuts proteins into small peptides, thus increasing the complexity of the samples and its subsequent analysis. Additionally this proteolysis step loses the post-translation modification information or amino acid mutations that may be driving a specific protein-protein interaction. Top-down methods avoid protein digestion and thus are used to directly evaluate the protein interactions or protein complexes. These two methodologies will bring the mass spectrometry and structural biology community a step closer to the realization of high-throughput structural biology for proteins and their interactions with other proteins and small molecules.

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

Post-translational modifications (PTMs) are important for regulating protein structure and function. Despite significant progress for PTM analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS), opportunities for new method development remain. The research presented in this dissertation promotes 193 nm ultraviolet photodissociation (UVPD) as an alternative activation technique for PTM analysis with specific utility for phosphorylated and sulfated peptides. A novel de novo sequencing method with applications for unbiased PTM discovery was developed utilizing Lys-N proteolysis, N-terminal imidazolinylation, and UVPD to direct fragmentation for the formation of N-terminal ions. The N-terminal a, b, and c ions generated by UVPD were differentiated from one another by characteristic mass shifts. Sets of triplet peaks were used to distinguish N-terminal ions from confounding C-terminal ions and improve the accuracy of de novo sequencing. UVPD was evaluated for the analysis of phosphopeptide cations and anions. Negative mode analysis was advantageous for the detection of casein peptides in high phosphorylation states, while positive mode proved more robust for global phosphoproteomic analysis of HeLa and HCC70 cell lysates. Compared to collisional activation, the depth of coverage was lower using UVPD yet more extensive fragmentation and improved phosphate retention on products ions was achieved. Phosphorylation mapping by LC-UVPD-MS was carried out in the C-terminal domain (CTD) of RNA polymerase II as a function of kinase treatment, ERK2 or TFIIH, and organism, yeast or fruit fly. Single phosphorylations on Ser2 or Ser5 in the consensus heptad, YSPTSPS, were observed across all experimental conditions. Analysis of the non-consensus fruit fly CTD revealed the significance of Tyr1 and Pro residues in the +1 position relative to Ser for phosphorylation to occur. For sulfated peptides, negative mode UVPD yielded a and x ions that largely retained the labile sulfate modification, facilitating peptide sequencing and PTM localization. With appropriate MS/MS tools established, the next step towards global sulfoproteomics was the development of enrichment methods. Weak anion exchange (WAX) was applied for this purpose. Following carbamylation to neutralize primary amines which otherwise repel the anion exchanger; improved WAX retention was observed for sulfopeptides relative to a complex mixture of unmodified bovine serum albumin peptides.

The aim of this book is to give a comprehensive description of the basic methods used in the ultraviolet spectroscopy of proteins, to discuss new trends and development of these methods, and to analyze their different applications in the study of various aspects of protein structure and dynamics. Ultraviolet spectroscopy is one of the oldest and most popular methods in the field of biochemistry and molecular biophysics. At present, it is difficult to imagine the biochemical laboratory without a recording spectrophotometer or spectrofluorimeter. There are several hundreds of publications directly devoted to protein ultraviolet spectroscopy and in a great number of studies UV spectroscopic methods are used for the structural analysis of different proteins. Meanwhile a unified description of the theoretical basis of the methods, experimental techniques, data analysis, and generalization of results obtained in solving the specific problems of protein structure are lacking. There are three reasons for which a monograph on ultraviolet spectroscopy is needed today. Firstly, there has been significant growth in facilities of experimental technique, its precision, and versatility associated with computer data analysts. This new technique is available to a wide circle of scientists engaged in the field of protein research. Most of them are not spectroscopists and, thus, there is a need for a conceivable and precise source of information on how to use this method and what kind of data it should provide.

The utility of 193 nm ultraviolet photodissociation (UVPD) and negative electron transfer dissociation (NETD) for the characterization of peptide anions was systematically evaluated. UVPD outperformed NETD in nearly all metrics; however, both methods provided complementary information to traditional collision induced dissociation (CID) of peptide cations in high throughput analyses. In order to enhance the performance of NETD, activated ion negative electron transfer dissociation (AI-NETD) methods were developed and characterized. The use of low-level infrared photoactivation or collisional activation during the NETD reaction period significantly improved peptide anion sequencing capabilities compared to NETD alone. Tyrosine deprotonation was shown to yield preferential electron detachment upon NETD or UVPD, resulting in N - C[alpha] bond cleavage N-terminal to the tyrosine residue. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages were regularly observed under high pH conditions. Transmission mode desorption electrospray ionization (TM-DESI) was coupled with 193 nm UVPD and CID for the rapid analysis and identification of protein digests. Comparative results are presented for TM-DESI-MS/CID and TM-DESI-MS/UVPD analyses of five proteolyzed model proteins. In some cases TM-DESI/UVPD outperformed TM-DESI-MS/CID due to the production of an extensive array of sequence ions and the ability to detect low m/z product ions. 193 nm UVPD was implemented in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa was achieved. The high-energy activation afforded by UVPD exhibited far less precursor ion charge state dependence than conventional methods, and the viability of 193 nm UVPD for high throughput top-down proteomics analyses was demonstrated for the less 30 kDa protein from a fractionated yeast cell lysate. The use of helium instead of nitrogen as the C-trap and HCD cell bath gas and trapping ions in the HCD cell prior to high resolution mass analysis significantly reduced the signal decay rate for large protein ions. As a result, monoclonal IgG1 antibody was isotopically resolved and mass accurately determined. A new high mass record for which accurate mass and isotopic resolution has been achieved (148,706.3391 Da ± 3.1 ppm) was established.