Microbes are found in all corners of the globe, and demonstrate extreme diversity in the roles they play within their niches. Syntrophic bacteria are a group of anaerobic microbes that break down fatty and aromatic acids, a process that forces them to survive at the edges of thermodynamic feasibility. Both the energetically limited state they exist, in combination with the large abundance of reactive acyl-CoA species present as metabolic intermediates, suggest a possibility of widespread lysine acylation, a common class of post-translational modification. We hypothesized that reactive acyl-CoA species would modify lysine residue with the corresponding acyl-lysine modification. Mass spectrometry was used to identify a wide array of lysine acylations in two species of bacteria, Syntrophus aciditrophicus and Syntrophomonas wolfei. As overly broad analysis could result in misidentifications, we identified diagnostic ion markers and developed methods utilizing these markers for detecting acyl-lysine modifications that could confidently identify even previously unknown modifications. Shotgun proteomics iii identified seven acylations in S. aciditrophicus, including those previously unidentified in the bacterial domain. Modifications identified were correlated with acyl-CoA intermediates in the cells. Acylations were enriched in pathways associated with aromatic acid degradation pathways. Mass spectrometry data acquired for the S. wolfei system revealed the same trends and suggested that lysine acylations changed quantitatively under different growth conditions. Together, the data support the “carbon stress” model of lysine acylation, whereby a buildup of reactive acyl-CoA species will non-enzymatically modify lysine residue in response to cellular conditions. The data also lays out a roadmap for future studies to determine the role these acylations play in regulating syntrophic cell metabolism.

Mass spectrometry (MS) has made significant contributions to protein and proteomics analysis during the past decades from its advantages of speed, sensitivity, specificity, and low sample consumption. While the proteomics field grows rapidly to identify thousands of proteins in a single analysis, “native” mass spectrometry, exploiting the unique features of electrospray ionization (ESI) for delivering large macromolecules to the mass spectrometer, has provided many potential exciting capabilities and applications to structural biology and biochemistry. It can analyze proteins in their native states, i.e., structures present in their native configurations from physiological pH solutions, with minimal sample preparation.

In this thesis, I describe the application of native ESI combined with top-down MS using electron capture dissociation (ECD) and ion mobility (IM) to characterize the molecular features of protein-ligand complexes. Binding and structural information can be comprehensively obtained from this experimental platform. Native ESI-MS alone provides molecular mass, stoichiometry, and binding affinity, all from a single analysis. We demonstrate that top-down MS, the fragmentation of intact proteins and protein complexes using MS, offers a powerful capability to elucidate the location of ligand binding on a protein’s structure and for probing the surface topology of proteins. Ion mobility mass spectrometry, a recently developed technique that yields information on the structural conformation of molecules, was used to reveal structural changes of proteins upon ligand binding.

My thesis focuses on several proteins, including α-synuclein (AS), which is a small protein related to Parkinson’s disease. AS is natively unfolded at physiological pH, which makes it difficult to study by standard methods such as X-ray crystallography or NMR. Using our mass spectrometry techniques, transition metal binding (copper, cobalt, and manganese) to AS that is associated with accelerating fibril formation was monitored. The binding of a small molecule amyloid inhibitor called molecular tweezer (MT or CLR01) on two model proteins important in neurodegenerative diseases, AS and superoxide dismutase (SOD1), was studied. Tandem mass spectrometry (MS/MS) techniques such as collisionally activated dissociation (CAD) along with ECD were used to characterize the sites of binding of small molecule ligands to proteins. Ion mobility mass spectrometry was implemented to reveal the conformational changes of AS upon metal binding. It was demonstrated that copper can induce the AS protein to collapse into a more compact state, which may provide a hint of the mechanisms behind amyloid fibrillation.

Additionally, two new methods to extend the application of top-down MS for protein structure characterization were developed. First, the same molecular tweezer molecule, which has a specificity to bind lysine residues, was used to probe surface residues of proteins. The lysines found to bind to the molecular tweezers identified by top-down MS correlates well with solvent accessibility values, suggesting that the MT compound can be applied as a molecular probe to pinpoint surface active lysine residues. Lastly, supplemental activation methods by ultraviolet and infrared laser irradiation prior to ECD was applied to assist disulfide bond cleavage of complex multiple intermolecular and intramolecular disulfide bond-containing proteins. Backbone bond cleavage from top-down MS was significantly increased when the disulfide bonds were cleaved, allowing more sequence information to be obtained. The new methods described in this thesis extend the applicability of mass spectrometry to provide a more complete picture of a protein’s structure.

Electrospray ionization (ESI) coupled to gas-phase electrophoretic mobility molecular analysis (GEMMA) is a soft ionization technique capable of characterizing large protein complexes. Particles are separated by their mobility in air and their size is determined as electrophoretic mobility diameter (EMD). This data can be converted to molecular weight information by modeling the particles as spheres and taking into account their average density. Here we adapt ESI-GEMMA towards the study of 1.5 MDa E2 protein cages and 9 MDa vault nanoparticles. Recombinant vaults with N- and C- terminal tags of varying mass were used as standards to calibrate the GEMMA data specifically for the study of vault complexes. We used this improved calibration to evaluate the composition and integrity of different vault preparations. Additionally, we quantified the capacity of vaults to be loaded with protein drugs and monitored the stability of these formulations over time.

Native mass spectrometry (MS) is a branch of MS analysis in which the structure of the target analytes of interest are kept intact and remaining in their “native” functional structure (as much as possible). This approach was made possible by the development of electrospray ionization (ESI), a soft ionization technique that does not fragment the target analyte during the ionization process while inducing multiple charging. The multiply charged biomolecules, in turn, can be subjected to fragmentation via collisional activation with a non-reactive gas such as nitrogen. This approach of combining native MS with fragmentation-based analysis, termed native topdown MS analysis, can be applied to large biomolecules such as membrane proteins to gain structural insights. Membrane proteins present unique challenges to conventional high-resolution structural techniques due to their hydrophobic nature. However, they are responsible for various physiological phenomena and account for 60% of known druggable targets in the cell. Thus, there is a need for an approach that can overcome issues with membrane protein analysis while complementing other biophysical techniques used to probe protein structure. Here, how native top-down MS can play this role is presented. The effects of non-ionic saccharide-based detergents, a commonly used class of detergents for membrane protein solubilization, on the resulting charge states of soluble proteins is investigated to gain insights into the mechanism of ESI. The MS-fragmentation patterns from collisionally activated dissociation of membrane proteins and membrane protein-lipid complexes are compared. How new insights into the lipid binding sites can be gained by detecting lipid-bound MS-fragments is presented. The result of the study indicates that native top-down MS analysis can provide unique structural insights for membrane proteins and their non-covalent interactions. When the analytical goal is to investigate the atomic composition of the target analyte, an ionization approach in which the sample is fully atomized before MS analysis is preferred instead. Inductively coupled plasma ionization, which atomizes and ionizes the sample via a plasma, can be coupled with MS analysis (ICP-MS) to quantify heavy metal contamination in complex samples. A protocol for ICP-MS analysis of commercial fish products for mercury contamination detection developed to aid an analytical chemistry class for instruction of undergraduate chemistry students is presented.

Neurotraumatic injury has long been a leading cause of death and disability worldwide. Recently, the debilitating long term effects of chronic, mild traumatic brain injuries (TBIs) have gained increased public attention. In order to protect individuals most at risk (e.g. military personnel and athletes) from such injuries, improved diagnostics in the form of a biomarker panel capable of rapidly and sensitively detecting mild TBIs are needed. Despite the large number of TBI biomarker studies in the literature, the development of a clinically relevant protein signature remains elusive.

In contrast to diseases with singular mechanistic dysfunction, neurotrauma is characterized by the disruption to multiple cellular pathways that contribute to the sequelae of secondary pathophysiology that determines patient outcome and recovery. This complexity has been a confounding factor in the identification of effective biomarkers. In an effort to circumvent this hurdle, our group implemented a central nervous system (CNS) specific cell injury model to examine preferentially released injury related proteins as candidate diagnostics.

Comparative analysis of a TBI CSF proteome and preferentially released proteins from our injury model revealed a panel of astroglial injury related candidate biomarkers including aldolase C (ALDOC), brain lipid binding protein (BLBP), glutamine synthetase (GS), astrocytic phosphoprotein PEA15 (PEA15), and glial fibrillary acidic protein (GFAP) and its trauma-generated breakdown products (BDPs). Immediate and robust release of ALDOC, BLBP, and PEA15 were associated more with acute cell wounding than cell death observed after biomechanical injury. In contrast, GFAP release correlated primarily with cell death. The sensitivity and selectivity of our biomarkers for neurotrauma were evaluated in human TBI and Yucatan swine spinal cord injury (SCI) CSF samples. Verification studies demonstrated the ability of our astroglial biomarker panel to differentiate injury from non-injury with elevated signals detectable an hour after injury. Additionally, differential CSF concentration kinetics were observed over a 1-week period post-injury indicative of a long diagnostic window. CSF concentration of biomarkers GFAP, ALDOC, and BLBP correlated strongly with the extent of tissue loss after SCI at 7 days. Taken together, our data demonstrates the successful application of proteomics to the identification and verification of new neurotrauma biomarkers that exhibit potential for not only detecting but defining injury severity.

Post-translational modification (PTM) of proteins is a conserved strategy used by organisms to efficiently control biological mechanisms, allowing rapid, adaptive cellular responses. PTMs can be categorized into two classes based on their biochemical origin: enzymatic and non-enzymatic. Non-enzymatic modifications arise from reactions between reactive electrophilic metabolites and nucleophilic or redox-sensitive amino acid side chains. Lysine acylation, a type of PTM, can occur spontaneously by intrinsically reactive metabolites, reactive acyl-CoA species (RACS). Metabolic pathways generate a variety of RACS that, in turn, produce equally diverse acyl-modifications. These PTMs regulate diverse biological mechanisms, deeply intersecting with cellular metabolism. Characterizing these PTMs comprehensively is important to understand their impact on metabolic regulation. While proteomic investigations have identified PTMs in bacteria, a majority of these studies focus on acetylation and not on other acylations formed by RACS.

Syntrophic bacteria are key players in microbial syntrophy, an important component of the global carbon cycle where different microbial species cooperate to decompose biopolymers into smaller molecules; e.g., hydrogen and methane. Syntrophic bacteria degrade fatty acid intermediates to hydrogen, formate, and CO2 and must cooperate with hydrogen consuming partners, methanogens, due to the reactions’ energetically unfavorable thermodynamics. As syntrophs live under thermodynamically challenging environments and its metabolic intermediates largely comprise of RACS, we hypothesized that these reactive metabolites lead to acyl-lysine modification and impact microbial metabolism. We utilized mass spectrometry (MS)-based proteomics to identify acyl-PTMs in syntrophic bacteria and developed MS methods to incorporate diagnostic marker ions for more confident identification of acyl-lysine. We characterized 10 types of acyl-modifications found amongst Syntrophus aciditrophicus and Syntrophomonas wolfei, including novel PTMs not reported in any other system. All modification types correspond directly to RACS produced in degradation pathways and a majority of modified proteins are involved in metabolism. The type and abundance of acyl-PTMs change with cultivation in different carbon substrates, linking protein acylation by RACS to shifts in cellular metabolism. We also characterized the proteomes of bacteria from other phyla, proteobacteria, identifying similar acyl-PTMs and raising the compelling possibility that non-enzymatic lysine acylation may be prevalent in microbial metabolism.

Nuclear and radiological terrorism is an on-going public health concern, but very few effective measures exist to assess the extent of the injury or counter the injuries from these potential attacks. In response to this need, the UCLA Center for Medical Countermeasures against Radiation (CMCR) has dedicated their research efforts on radiation biodosimetry and drug development. On the diagnostic side, existing biodosimetry is only able to provide a crude estimate of radiation exposure dose. More effective diagnostic tools are needed to confirm exposure and predict tissue-specific radiation injury progression. Towards this end, we aimed to develop protein biomarkers that can assess organ-specific radiation damage. Utilizing quantitative mass spectrometry (MS)-based proteomics, we performed discovery experiments to identify proteins that have desirable biomarker characteristics. In addition, we evaluated a set of hypothesized biomarker candidates as part of antioxidant response using a targeted MS method. On the treatment side, very few medical products are available to mitigate radiation-induced injury. In fact, only three radiomitigators, through drug repurposing, have been approved by the FDA for treatment of hematopoietic acute radiation syndrome (H-ARS). The UCLA CMCR has recently identified a novel group of small molecules from high throughput screening (HTS) for inhibitors of radiation-induced apoptosis. The lead compound dramatically decreases mortality from H-ARS in mice. To elucidate the mechanism of action for the lead compound, we utilized an emerging target identification approach based on thermal stability shift upon ligand binding (i.e. thermal proteome profiling or TPP). Data from TPP experiments proposed hypothetical targets for the lead compound, which can later be validated by protein-ligand binding studies and other means.

Protein identification by top-down mass spectrometry based methods yield intact mass of the proteins and indicate the presence of post-translational modifications (PTMs) and/or isoforms. Currently, the methods employed for top-down protein identification are performed using instruments with dual mass analyzers and are based on fragmenting isolated charge states, which greatly reduces the duty cycle of the instrument. High throughput top-down methods are required for protein identification in complex sample mixtures. We demonstrate the capability to perform intact protein identifications in a single-stage time-of-flight mass spectrometer during protein elution from a liquid chromatography (LC) column. In addition, we have developed a new data-independent fragmentation method known as `Continuous Accumulation of Selected Ions-Collisionally Activated Dissociation' (CASI-CAD) to fragment multiple charge states of the protein simultaneously for the purpose of identification in the LC timescale. CASI-CAD is performed without any precursor selection and thus, the duty-cycle of the instrument is not lowered. Both these methods unambiguously identified all the proteins in the human proteasome complex used for method development. The presence of PTMs and N-terminal modifications were also characterized for the proteins in this complex.

Supercharging reagents are known for their ability to enhance the multiple charging of proteins during electrospray ionization (ESI). This improves the mass measurement accuracy and fragmentation efficiency of proteins during ESI-MS. Currently, the mechanism behind supercharging is unknown. We have analyzed different supercharging reagents under a variety of solvent conditions to probe the mechanisms behind supercharging. In addition, the supercharging ability of sulfolane was utilized for proteins eluting from a column by adding the reagent to the LC solvents. Furthermore, reagent introduction in the vapor phase increased the signal intensity for intact proteins eluting from a column when compared to experiments performed without the reagent. These methods presented here are efficient top-down means to address complex samples in the chromatographic timescale.

Transcriptomics is often touted to be the silver bullet to determining the protein levels within a microorganism when comparing such proteins under two or more different biological conditions. However, designating the transcriptome as a surrogate for protein abundance can be misleading and impractical when determining the effect of environmental stresses on the phenotype of an organism. While the transcriptome is significant for identifying genes that are linked to, or act as co-regulators in the response to stressors, it is best to consider proteomic analyses, in addition to transcriptional profiling, as a complementary tool in order to determine the effect of different environmental factors on the fitness and phenotype of the microorganism in question. Methanosarcina mazei are mesophilic archaea microorganisms that grow under anaerobic conditions and produce methane via an energy-conservation process, known as methanogenesis, which is the terminal step in the degradation of organic matter during the carbon cycle. While the Methanosarcina are versatile in their ability to grow on numerous substrates, such as methanol and other compounds such as monomethylamine (MMA), dimethylamine (DMA), and trimethylamine (TMA), it is more thermodynamically favorable for the cells to grow on methanol (∆G’o = -106 kJ/ mol methane) than the methylamines (∆G’o = -77 kJ/ mol methane). Therefore, the lower free energy change in using the methylamines necessitates M. mazei cells to have an efficient energy-conserving system to deal with the thermodynamic limitations. Consequently absolute quantitation using a label-free proteomics strategy was applied to determine the intercelluar protein abundances of the Methanosarcina mazei proteome with different methylotrophic substrates (i.e., methanol, MMA, DMA, and TMA). Specifically, the aims of this project are to use quantitative proteomics to: (1) establish how M. mazei uses enzymes involved in methanogenesis to convert chemical energy into biomass; (2) determine how methanogenic process depends upon substrate type during the mid-log and stationary phases of growth; (3) determine whether different salt concentrations affect the glycosylation pattern of surface of the protein, and in turn methanogenesis; (4) understand the effect of methylotrophic substrate availability on a different methanogen, M. barkeri. At least 40% of all of the proteins were found to be methyltransferases and methylcoenzyme-M reductases in M. mazei cells grown under mid-log and stationary phase conditions. Furthermore, the protein abundances varied in a substrate-specific manner, indicating that the M. mazei cells may have evolved to be prepared for a potential carbon source switch.

Proteomics has flourished as a result of major advances in computational power, data storage technologies, and network architecture, all of which enable the acquisition, handling, and processing of enormous data sets. As mass spectrometers continue to break resolution and sensitivity barriers, the limitations of qualitative and quantitative proteomic experiments will no longer result from instrumentation constraints, but rather from the quality and availability of robust sample preparation methods and post-processing algorithms. My dissertation focuses on the development of technologies in experimental proteomic workflows that precede and that follow mass spectrometric analysis - areas that are integral to the future of proteomics.

Enhanced Filter Aided Sample Preparation (eFASP) incorporates Tween-20 for plastic passivation, and n-octylglucoside and deoxycholic acid for digestion enhancement. In E. coli, eFASP reduces sample losses associated with normal FASP by almost 300%, and significantly increases hydrophobic protein representation when compared to normal FASP and to a routine precipitation-based sample preparation. eFASP is validated in the analysis of the ultra-stable proteinaceous sheath of Methanospirillum hungatei. eFASP and label-free quantitative analysis revealed a 40.7 kDa polypeptide (Q2FRN9) encoded by Mhun_2271 as the predominant sheath component by 10-fold.

eFASP is combined with sample fractionation methods and software tools for the quantitative analysis of bacterial and human samples. Hydrophilic interaction chromatography (HILIC) enables small-scale fractionation for less complex samples: Syntrophus aciditrophicus, Anaerobaculum hydrogeniformans, and melanoma-derived exosomes. S. aciditrophicus and A. hydrogeniformans are profiled for proteome shifts resulting changes in carbon source. Treatment of melanoma cells with B-Raf inhibitor, PLX-4720, results in the packaging of two tyrosine kinases, VEGFR and PDGFR-B, into secreted exosomes. Upregulation of these receptor tyrosine kinases has been found to be a result of the release of feedback inhibition following treatment with MAPK targeted therapeutics.

Complex whole-cell proteome analysis is effected by high-resolution electrostatic-repulsion hydrophilic interaction chromatography (ERLIC) fractionation of eFASP-processed samples. eFASP-ERLIC facilitated the quantitative analysis of normal and ATM-deficient human lymphoblastoid cell lines (LCLs) exposed to ionizing radiation (IR) and to YEL2, a potent radiomitigator. Exposure to IR resulted in the durable upregulation of NRF2-oxidative stress response components in Normal LCLs, while thrombin signaling and pro-survival proteins were upregulated in ATM-deficient LCLs. YEL2 treatment upregulated DNA repair components and enriched pro-survival pathways, which implicate the involvement of multiple processes in YEL2-mediated radioresistance.