• Advances in Mass Spectrometric Structural Biology Techniques for Pattern Recognition Receptor Ligands of Microbial Origin

      Oyler, Benjamin; Goodlett, David Robinson, 1960- (2018)
      Pattern recognition receptors (PRR) are the innate immune system’s first-line sentinels for distinguishing “self” from “non-self.” Many molecules found in the bacterial cell wall are PRR ligands, including lipopolysaccharide (LPS), cardiolipin (CL), and peptidoglycan (PGN). Molecular structural biology techniques are essential for determining both basic cell biology and host-bacteria interactions through ligand-receptor binding mechanisms. Recent interest in designer PRR ligands or PRR ligand mimetics for use in drug discovery pipelines have given this research more translational value as well. Mass spectrometry (MS) has the unique capability to derive primary structures of ions as well as monitor many different ions in complex mixtures. Several different advances in PRR ligand structure analysis were achieved in this dissertation. First, chemical structure of an LPS-derived vaccine was determined using a top down tandem MS approach. Several different instrumental configurations and methods were employed to demonstrate complementarity of data and broad applicability of the approach. Second, CL from a newly discovered Actinomycete marine sponge symbiont was analyzed and compared to CL from a terrestrial Firmicute to generate hypotheses about host-bacterium interactions. This was the first molecular analysis of any secondary metabolites from this species of bacteria. Third, PGN subunits (muropeptides) from Rickettsia typhi were analyzed in a data dependent global LC-tandem MS approach. This was the first example of PGN structure discovery for R. typhi and the first example of this approach applied to PGN structure elucidation for any Rickettsiae species. All of these developments will help to advance PRR-ligand interaction research – an emerging and promising field for development of novel disease treatment and prevention approaches. Modulation of the innate immune response to bacterial insult is a challenging task without a clear understanding of underlying molecular mechanisms and how they might be manipulated by medicine. One key step in this process is development of sensitive and specific chemical analysis methods fit to acquire unequivocally interpretable data. While all of the methods described herein were applied to specific biological problems, their applicability to other scientific questions is broad.
    • Applications of Quantitative Proteomics and Phosphoproteomics to Study the Development of Resistance to Targeted Therapy in Cancer

      Awasthi, Shivangi; Shapiro, Paul, Ph.D.; Goodlett, David Robinson, 1960-; Guha, Udayan (2018)
      Targeted inhibition of protein kinases is a major approach to treat cancer. However, the effectiveness of kinase inhibitors is limited due to intrinsic and acquired resistance mechanisms that promote the progression and survival of cancer cells. The objective of this dissertation is to use liquid chromatography coupled to mass spectrometry (LC MS) based quantitative proteomics to identify potential biomarkers of resistance and response to molecularly targeted therapies in cutaneous melanoma and lung adenocarcinoma in vitro. For the first part of this thesis, I conducted a proteomic analysis of the acquired drug resistance to extracellular signal-regulated kinase (ERK1/2) pathway inhibitors in a melanoma cell line model. A combination of immunoblot assays, global label-free bottom-up proteomics, phosphoproteomics and pathway analysis was used to characterize the differential protein expression in drug resistant melanoma cells. Examination of the quantitative data pointed to an invasive and metastatic phenotypic signature in the resistant cells. We also identified and verified the overexpression of β-catenin and Caveolin-1 (CAV-1) in MEK1/2 and ERK1/2 inhibitor resistant cells. These findings suggest that these proteins have a role in the development of resistance and may represent novel targets for co-therapy. For the second part of this thesis, I have utilized a multiple reaction monitoring (MRM) based targeted proteomic technique to verify previously identified potential biomarkers of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) response in lung adenocarcinoma. Published global phosphoproteomic data were used to select a list of phosphotyrosine peptides (pY) and MRM based relative and absolute quantitative methods were developed to measure their expression in TKI sensitive and resistant lung adenocarcinoma cells. Modified immuno-MRM assays were optimized using heavy labelled synthetic peptide standards which identified the targets with good reproducibility and repeatability. The results indicated that of the 11 chosen sites, EGFR-pY1197 can be used as potential biomarker of EGFR TKI sensitivity, regardless of the EGFR TKI used. Overall these data advance our understanding of the mechanisms of targeted therapy resistance and highlight candidate biomarkers of resistance and sensitivity.
    • Mass Spectrometry based structural analysis and systems immunoproteomics strategies deduce specifics of host-pathogen interactions

      Khan, Mohd M.; Goodlett, David Robinson, 1960- (2018)
      The innate immune system is the first line of defense against pathogens. Pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs) sense and sample pathogen-associated molecular patterns (PAMPs). On the host myeloid cell surface, the proinflammatory Gram-negative bacterial outer membrane component lipopolysaccharide (LPS, also known as endotoxin) activates the innate immune system via TLR4. Intracellularly, LPS is detected by the noncanonical inflammasome through caspase4/5/11. In the present work, mass spectrometry (MS)-based top-down structural analysis of LPS uncovered major determinants of molecular pathogenesis, and MS-based systems immunoproteomics elucidated specific features of the immune response against endotoxin. We used targeted proteomics to profile the host response to the pathogens Escherichia coli, Staphylococcus aureus, and Burkholderia cenocepacia, and we discovered significant temporal changes in the macrophage secretome. Additionally, we identified global changes in protein secretion in TLR4- and caspase11- stimulated macrophages. Finally, we observed bacterial proteomic rewiring within the biofilm forms of Burkholderia, possibly explaining the observed lowering in sensitivity to antibiotics.
    • Rapid Diagnosis of Microbial Infection via Mass Spectrometric Phenotyping

      Liang, Tao; Goodlett, David Robinson, 1960-; 0000-0001-9883-3999 (2018)
      Microbial infection is a perpetual public threat, causing more than 15 million annual deaths worldwide. Clinical microbiology laboratories currently rely on bacterial culture-based methods for microbial diagnosis to identify causative pathogens, which is time-consuming and labor-intensive. This drives the development of novel diagnostics to identify pathogens accurately and more rapidly. Mass spectrometry (MS) now plays a vital role in clinical diagnosis due to its high accuracy, high specificity, rapidity and high- throughput capability. In this thesis, we present a multi-faceted mass spectrometry approach for rapid microbial infection diagnosis. We first used a novel sample transfer technique, surface acoustic wave nebulization (SAWN) for bacterial membrane lipid analysis, specifically lipid A. Analytical performance of different SAWN chips was characterized, and the optimized SAWN chip was used for bacterial phenotyping. Results showed that lipid A mass spectra from different bacterial species can be differentiated by dot product analysis, in turn, demonstrating feasibility of using SAWN for rapid bacterial identification. We next developed a rapid sodium acetate (SA) based method for lipid extraction, which greatly improved our lipid-based library for pathogen identification by reducing the process time to less than an hour. Importantly, the novel SA method maintained the key components of the reported lipid library method for bacterial identification. Namely, these were the ability to detect 1) antibiotic resistance, 2) microbes direct from biological fluids without culture, and 3) single microbes in polymicrobial samples. This platform can be a complementary approach to the commercialized protein-based systems to improve patient outcomes. The last objective of this thesis is to understand the proteome change in response to lipopolysaccharide stimulus in the context of sepsis, which will facilitate the discovery of new biomarkers for sepsis diagnosis. Shotgun label-free quantification proteomics results showed that 27 new sepsis-related proteins were found among 182 significantly changed proteins in the septic mouse group. A longitudinal, but not pair-wise, data analysis strategy overcame inherent heterogeneity detected twice as many significant changes using each mouse's data as its own control sample. Overall, the advances made in this thesis have broad implications in MS-based rapid diagnosis of microbial infections.