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Inexpensive label-free detection of biomarker panels in serum could revolutionize earlycancer diagnosis and treatment. Such detection capabilities may be possible with dynamicnanochannels in conjunction with electrical impedance measurement. In Dr. Greg Nordin's lab I designed, fabricated and tested several iterations of these sensors with polydimethyl-siloxane microfluidics. The final design yielded a dynamic nanochannel array sensor thatshowed a 140% impedance change when exposed to 14µM bovine serum albumin in phos-phate buffered saline. For the geometry and noise limits of the tested device, simulationsindicated that a minimum detectable concentration of 20pM with specifically bound strep-tavidin should be possible. However, the polydimethylsiloxane approach is also shown to beproblematic in meeting the trade-offs required for a practical device. Consequently, alter-native materials and designs are suggested to reduce the minimum detectable concentrationto the high femtomolar range, which would be attractive for detection of many medicalbiomarkers.

The sea squirts Ciona intestinalis and C. savignyi have disparate distribution patterns, which may result from differences in their thermal tolerance limits. Because C. intestinalis, an almost cosmopolitan species, has a more widespread distribution, it is thought that it is better adapted to endure a wide range of temperatures. In order to compare the heat stress response between these two congeners, we studied global changes in protein expression, using a proteomics approach. To characterize the response to extreme heat stress, animals of both species were exposed to temperatures of 22°C, 25°C, and 28°C for 6 h, and then were left to recover at a control temperature (13°C) for 16 h. An additional experiment was conducted to assess the effect of mild-to-moderate heat stress including a 6 h exposure to temperatures of 18°C, 20°C, and 23°C, and a 16 h recovery at a control temperature (16°C). A quantitative analysis, using 2D gel electrophoresis and gel-image analysis, showed that in the high heat stress (HHS) experiment, 15% and 18% of the all protein spots detected demonstrated changes in expression in C. intestinalis and C. savignyi, respectively. In the low heat stress (LHS) experiment, 4% of the total number of proteins detected changed significantly in both C. intestinalis and C. savignyi. Using matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight mass spectrometry, we were able to identify proteins with a 65-100% success rate, depending on species. Our results indicate that C. intestinalis maintains higher baseline levels of molecular chaperones and launches a quicker response to thermal stress than C. savignyi, suggesting it may be the more thermally tolerant of the two. In addition, actins, tubulins, and ATP-synthase F1 β-subunits were the most susceptible to proteolytic degradation, which may indicate that they have relatively higher thermal sensitivities.

The ascidian species Ciona savignyi and C. intestinalis are invasive species but show interspecific differences in their population response to hypo-saline stress associated with heavy winter-run off events that are predicted to become more frequent due to climate change. Despite an almost world-wide distribution, C. intestinalis seems to be more susceptible to hypo-saline stress than the geographically more limited C. savignyi. Given that the genomes of both species are fully sequenced, we were able to compare their proteomic response to both acute and chronic salinity to characterize the mechanisms that are responsible for setting tolerance limits to hyposaline conditions in these two congeneric species. For the acute hypo-saline stress experiment, we exposed each species to decreasing salinities, 100%, 85% and 70% full-strength seawater, for 6 hours followed by a 4-hour recovery at 100%. In the chronic salinity stress experiment, each species was kept at 100% or 85% with individuals removed for analysis during a 16-day time course. Organisms were dissected to remove the tunic, and 2D SDS-PAGE was performed to separate proteins and characterize changes in protein abundances. In the acute experiment, we determined 5% and 19% of the proteins to be significantly changing abundance in C. savignyi and C. intestinalis, respectively, due to the treatment effect. For both species in the chronic experiment, we determined over 40% of the proteins to be significantly changing abundance given the treatment, time, or interaction effect. Analysis of these proteins with MALDI TOF-TOF mass spectrometry has identified numerous proteins implicated in cellular stress responses, including energy metabolism (glycolysis, ATP & NADH production), cytoskeletal restructuring, and protein turnover, providing insights into the intense cellular restructuring that occurs following hypo-saline exposure.

Increases in seawater temperatures have imposed physiological constraints which are partially thought to contribute to recently observed shifts in biogeographic distribution among closely related intertidal ectotherms. For instance, Mytilus galloprovincialis an introduced warm-adapted species from the Mediterranean, has displaced the native cold-adapted congener, M. trossulus, over large latitudinal expanses off the California coast. Several comparative physiological studies have revealed interspecific differences in thermal tolerance, including variation in aerobic metabolism and gape behavior, which suggest the invasive congener is better adapted to acclimate to increasing seawater conditions as predicted due to climate change. However, current analyses seek to discover the cellular process which contribute to thermal plasticity at the level of the whole organism in response to temperature stress. Since proteins represent the primary molecular machinery capable of responding to thermal stress, we quantified the proteomic response of the adductor muscles (AM) of M. galloprovincialis and M. trossulus to acute heat stress. After acclimation to 13°C, we exposed mussels to 24°C, 28°C and 32 °C (at a heating rate of 6C/h), kept mussels at the temperature for 1 h and then added a 24-h recovery period. Posterior adductor muscle samples were then excised and utilized for proteomic analysis. We were able to detect 273 protein spots within M. galloprovincialis and 286 protein spots within M. trossulus. Roughly 33% of these protein spots exhibited significant changes in abundance in response to heat stress within M. trossulus as compared to only 19% in M. galloprovincialis. In both data sets, most proteins changing abundance are part of the cytoskeleton or proteins controlling actin thin filament dynamics and stress fiber formation. Specifically, M. galloprovincialis increased the abundance of proteins involved in thin filament stabilization and cytoskeletal maintenance. In contrast, M. trossulus increased proteins involved in thin filament destabilization and filament turnover. In addition, only M. trossulus increased proteins involved in the cellular stress response at the highest temperature, suggesting its AM proteome is more thermolabile. In return, our results suggest that cytoskeletal architecture is more thermally stable in M. galloprovincialis. The differences in the proteomic responses suggest that M. galloprovincialis is capable of protecting itself from heat stress through valve closure at a higher temperature due to the increase in actin stabilizing proteins.

Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins.

Cryptosporidium parvum and Toxoplasma gondii are obligate intracellular parasites transmitted by ingestion of resilient walled structures called oocysts. Infection is self-limiting in adults with normal immune systems. However, severe disease can occur in immunocompromised individuals, or those without cellular immunity. Cryptosporidium is a leading cause of infant mortality in developing countries, due to diarrhea. There are no human vaccines and no broad effective drug treatments. Several vaccine candidates have been described: the glycoproteins Gp900, Gp40, and Gp15 and the protein Cp23, the immuno-dominant-antigen. Details about modifications to these proteins have not previously been reported. Using mass spectrometry, we identified 16 Cryptosporidium N-glycosylated proteins, including Gp900 and a possible oocyst wall protein. The observed N-glycan structures exhibited only two compositions: HexNAc2Hex5 and HexNAc2Hex6; these glycoforms had a single extended arm. The simplicity of Cryptosporidium N-glycans contrasts with the complexity of host N-glycans. Four heavily O-glycosylated proteins included Gp900, Gp40, Gp15, and a novel mucin-like protein, Gp20. Single O-HexNAc residues modified Ser/Thr in low density regions of Gp15 and Gp900, while attachment of O-HexNAc residues on tandem Ser/Thr repeats of Gp20 and Gp40 approached saturation. Identification of N-acetylgalactosamine (GalNAc) as the HexNAc released from proteins suggests that most Cryptosporidium O-glycans resemble the immunogenic Tn antigen (O-GalNAc). The immunodominant antigen Cp23, while not glycosylated, was discovered to be N-myristoylated and S-palmitoylated on the first and second residues, respectively. This is the first identification in Cryptosporidium of these modifications. Information about the N-glycans, O-glycans, and lipid modifications may be useful for design of better serodiagnostic reagents and more effective vaccines. To date, there are no vaccines against Toxoplasma infection, and the only available pharmaceutical therapies are expensive. In the second study, a novel O-fucose modification was discovered on nuclear pore-associated proteins including nucleoporins. This observation has profound implications on how the organism may regulate trafficking in/out of the nucleus by employing a system parallel to that described for O- linked N-acetylglucosamine in other organisms. In summary, the new details regarding the vaccine candidates of Cryptosporidium and the discovery of the novel O-fucose modifications in T. gondii provide information that could prove useful for development of effective drugs and vaccines. / 2018-11-01T00:00:00Z

Parkinson’s disease (PD) is a neurological disorder characterized by the lack of functional dopaminergic neurons in the nigrostriatal pathway in the brain. Current therapeutic strategies for the disease provide temporary symptomatic relief. Gene therapy has the potential to improve dopamine production in Parkinson’s disease patients. Adeno-associated viruses (AAV) are the vectors of choice in gene therapy for PD, due to their well-characterized safety and efficacy profiles, with all primary receptors being glycans. The problem with using AAV in PD gene therapy is that the aged brain is resistant to transduction of the virus, while PD primarily occurs with age. Thus, in Aim 1 we characterize the age-related changes in glycan receptors in the nigrostriatal pathway as a baseline to address current challenges in gene delivery in Parkinson’s disease. To make these measurements from specific regions of tissue, we develop a platform that incorporates on-slide digestion followed by LC-MS/MS for integrated glycomics and proteomics. Further, we apply this to understand aging- and PD-related changes in the human pre-frontal cortex in Aims 2 and 3, to understand normal and pathological aging processes as well as integrate this information with transcriptomics data, to assess risk factors that may contribute to Parkinson’s disease. Finally, we also apply the method to investigate cancer premalignancy and heterogeneity. Our on-slide method, used herein to study aging, Parkinson’s disease and cancer, can be applied to any precious biopsy specimens to enable glycomic and proteomic profiling in diverse diseases, and thus may have a broad impact on biomedical research.

Cellular Thermal Shift Assay (CETSA) is a method of identifying protein-drug interactions by monitoring changes in protein thermal stability. CETSA is traditionally performed by using Western Blotting to examine the thermal stability shifts of a single protein of interest. By combining CETSA with Mass Spectrometry the shifts in thermal stability can be examined for an entire proteome in a single experiment in a technique known as CETSA-MS or Thermal Proteome Profiling (TPP). This can be used to identify targets of a compound of interest in order to further understand the compounds mechanism of interest, potentially making CETSA a powerful tool for target discovery. Here we attempt to develop a protocol by which CETSA can be used as a drug target discovery tool. Our work has allowed us to create a protocol that can reliably identify soluble drug targets. Our results demonstrate the capacity of CETSA to screen multiple compounds as well as to perform more in depth dose response studies, and highlight how future improvements could be made to the protocol

Traditional proteomics studies can measure many protein biomarkers simultaneously from a single patient-derived sample, promising the possibility of syndromic diagnoses of multiple diseases sharing common symptoms. However, precious cellular-level information is lost in conventional bulk-scale studies that measure tissues comprising many types of cells. As single cells are the building blocks of organisms and are easier to biopsy than traditional bulk samples, performing proteomics on a single-cell level would benefit clinicians and patients. Single-cell proteomics, combined with mass spectrometry imaging, can be used to analyze cells in their microenvironment, preserving spatial information. We have previously used laser-capture microdissection to isolate single motor neurons from tissue and analyze them in our single-cell proteomics platform. However, our sampled population of cells was necessarily limited by the low throughput of the measurement platform, and by the sensitivity of our liquid chromatography-mass spectrometry system to debris introduced in the laser-capture microdissection isolation workflow. In the work described in this dissertation, we dramatically improved the throughput of single-cell proteomics, created a method for removing insoluble debris that clogged our liquid chromatography-mass spectrometry system, and developed a high-performance, low-cost method for nanoflow gradient formation. Together, these methodologies will increase the depth of information and the number of biological replicates that can measured in single-cell proteomics. We hope that these technologies will be applied to future liquid chromatography systems to enable large scale single-cell proteomics studies of tissues. This will reveal the cellular origins of disease on a multimolecular level, while keeping important spatial information. Thus, we expect the technologies and ideas developed here to play a key role in understanding the cellular proteomics in biomedical and clinical settings.