A global analysis of the human proteome demonstrates that there are ~5500 tryptic fragments that contain four cysteines in close proximity. Elucidating whether they form disulfide bonds in vivo under different conditions is particularly important because cysteines are known to be a vital cellular redox sensor as well as a catalytic site for important biochemical reactions. However, currently there are no methods that can resolve disulfide patterns in closely-packed cysteine residues from a complex sample. In order to address this problem, we have developed a novel mass-spectrometry-based method to identify the different disulfide bonding patterns possible, using SNAP25B cysteine-rich region as a test case. Unlike traditional proteomics, this method uses non-reduced sample preparation, thus preserving intact disulfide bonds. It relies on collision-induced dissociation (CID) to cause double-backbone and heterolytic disulfide-bond cleavage and compares this to the theoretical MS/MS spectra. CID in an ion trap gives robust detection of double backbone cleavages and heterolytic disulfide-bond cleavages. Here, we report, for the first time, identification of all three disulfide patterns for double-disulfide species of SNAP25B using collision-induced dissociation.

No description available.

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

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.

The initial steps involved in the assembly of normally soluble proteins into amyloid fibrils remain unclear, yet over 20 human diseases are associated with proteins that aggregate in this manner. Protein surface modification is a potential means of mapping the interaction sites in early oligomers that precede amyloid formation. This dissertation focuses on the use of covalent labeling combined with mass spectrometry to elucidate the structural features of Cu(II)-induced β-2-microglobulin (β2m) amyloid formation. An improved covalent modification and MS-based approach for protein surface mapping has been developed to address the need for a reliable approach that ensures protein structural integrity during labeling experiments and provides readily detectable modifications. This approach involves measuring the kinetics of the modification reactions and allows any local perturbations caused by the covalent label to be readily identified and avoided. This MS-based method has been used to study human β2m, a monomeric protein that has been shown to aggregate into amyloid fibrils in dialysis patients leading to dialysis-related amyloidosis. Under conditions that lead to β2m amyloid formation, reactions of β2m with three complementary covalent labels have been used to identify the Cu(II) binding site, metal-induced conformational changes, and the oligomeric interfaces. Results confirm that Cu(II) binds to His31 and the N-terminal amine. Binding to these residues causes several structural changes in the N-terminal region and ABED β-sheet which likely enables formation of oligomeric intermediates. The covalent labeling data indicate that the pre-amyloid β2m dimer has an interface that involves the antiparallel arrangement of ABED sheets from two monomers. Moreover, our covalent labeling data allowed us to develop a model for the tetramer in which the interface is mediated by interactions between D strands of one dimer unit and the G strands of another dimer unit. Lastly, the selective covalent modification approach has been used to delineate the structural changes in β2m after interaction with Cu(II), Ni(II), and Zn(II) and their effect on its aggregation. Our covalent labeling data indicates that the unique effect of Cu(II) appears to be caused by the site at which the metal binds the protein and the conformational changes it induces.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24187号 / 医博第4881号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 貴浩, 教授 滝田 順子, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No abstract provided. / Thesis / Doctor of Philosophy (PhD) / Scope and contents: The principal aim of the present investigation is to extend our knowledge of the merits and demerits of the mass spectroscopic method of solid analysis. This dissertation provides information concerning relative ionization efficiencies for several elements and factors which effect these efficiencies. Secondary purposes of this work are 1) to make an independent determination of the temperature of the vacuum spark, 2) to resolve the current discrepancy concerning the value of the C3+/C2+ ratio from graphite electrodes in a spark and, finally, 3) to provide an accurate value for the Ni58/Ni60 abundance ratio.