Glycosylation, the addition of one or more carbohydrates molecules to a protein, is crucial for many cellular processes. Aberrant glycosylation is a key marker for various diseases such as cancer and rheumatoid arthritis. It has also recently been discovered that glycosylation is important in the ability of the Human Immunodeficiency Virus (HIV) to evade recognition by the immune system. Given the importance of glycosylation in disease, major efforts are underway in life science research to investigate the glycome, the entire glycosylation profile of an organelle, cell or tissue type. To date, little bioinformatics research has been performed in glycomics due to the complexity of glycan structures and the low throughput of carbohydrate analysis. Recent advances in mass spectrometry (MS) have greatly facilitated the analysis of the glycome. Increasingly, this technology is preferred over traditional methods of carbohydrate analysis which are often laborious and unsuitable for low abundance glycoproteins. When subject to mass spectrometry with collision-induced dissociation, glycopeptides produce characteristic MS/MS spectra that can be detected by visual inspection. However, given the high volume of data output from proteome studies today, manually searching for glycopeptides is an impractical task. In this thesis, we present a tool to automate the identification of glycopeptide spectra from MS/MS data. Further, we discuss some methodologies to automate the elucidation of the structure of the carbohydrate moiety of glycopeptides by adapting traditional MS/MS ion searching techniques employed in peptide sequence determination. MS/MS ion searching, a common technique in proteomics, aims to interpret MS/MS spectra by correlating structures from a database to the patterns represented in the spectrum. The tool was tested on high throughput proteomics data and was shown to identify 97% of all glycopeptides present in the test data. Further, the tool assigned correct carbohydrate structures to many of these glycopeptide MS/MS spectra. Applications of the tool in a proteomics environment for the analysis of glycopeptide expression in cancer tissue are also be presented.

Glycosylation, the addition of one or more carbohydrates molecules to a protein, is crucial for many cellular processes. Aberrant glycosylation is a key marker for various diseases such as cancer and rheumatoid arthritis. It has also recently been discovered that glycosylation is important in the ability of the Human Immunodeficiency Virus (HIV) to evade recognition by the immune system. Given the importance of glycosylation in disease, major efforts are underway in life science research to investigate the glycome, the entire glycosylation profile of an organelle, cell or tissue type. To date, little bioinformatics research has been performed in glycomics due to the complexity of glycan structures and the low throughput of carbohydrate analysis. Recent advances in mass spectrometry (MS) have greatly facilitated the analysis of the glycome. Increasingly, this technology is preferred over traditional methods of carbohydrate analysis which are often laborious and unsuitable for low abundance glycoproteins. When subject to mass spectrometry with collision-induced dissociation, glycopeptides produce characteristic MS/MS spectra that can be detected by visual inspection. However, given the high volume of data output from proteome studies today, manually searching for glycopeptides is an impractical task. In this thesis, we present a tool to automate the identification of glycopeptide spectra from MS/MS data. Further, we discuss some methodologies to automate the elucidation of the structure of the carbohydrate moiety of glycopeptides by adapting traditional MS/MS ion searching techniques employed in peptide sequence determination. MS/MS ion searching, a common technique in proteomics, aims to interpret MS/MS spectra by correlating structures from a database to the patterns represented in the spectrum. The tool was tested on high throughput proteomics data and was shown to identify 97% of all glycopeptides present in the test data. Further, the tool assigned correct carbohydrate structures to many of these glycopeptide MS/MS spectra. Applications of the tool in a proteomics environment for the analysis of glycopeptide expression in cancer tissue are also be presented.

Orientador: Gonçalo Amarante Guimarães Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T09:47:13Z (GMT). No. of bitstreams: 1 Salazar_MarcelaMendes_D.pdf: 4370521 bytes, checksum: 99869d0c9655d47c4e3b2c29de58a274 (MD5) Previous issue date: 2012 / Resumo: A celulose é o polímero mais abundante do planeta e está presente principalmente na parede secundária de células vegetais maduras. O conhecimento dos mecanismos moleculares envolvidos na sua biossíntese, bem como a regulação deste processo é recente e ainda elementar, apesar da sua grande importância. O Eucalyptus é o gênero florestal mais plantado no mundo, especialmente por ser matéria-prima para fabricação de celulose. Investimentos em pesquisas e desenvolvimento estão sendo realizados pelo setor florestal, no intuito de aumentar os ganhos de produtividade de celulose através da pesquisa na área da biologia molecular. Os dados analisados desmostram que a expressão gênica das espécies estudadas (E. globulus, E. grandis e E. urophylla) difere em genes essenciais para a formação dos compostos da parede celular e por fim da madeira. Os dados moleculares são condizentes com dados diferenciais aqui encontrados em relação à composição dos açúcares da parede. Os resultados gerados pela análise de composição da parede por diferentes técnicas, incluindo a recentemente desenvolvida "Perfil Glicômico" mostram uma grande diversidade e quantidade dos carboidratos da parede celular diferentemente distribuídos no xilema das espécies Eucalyptus globulus, grandis e urophylla, bem como em folha. Tais resultados apresentam um grande avanço para o entendimento da composição das paredes celulares de tais espécies. Assim, o objetivo do presente trabalho foi correlacionar os dados moleculares, com dados highthroughput do Perfil Glicômico para entender a composição e arquitetura da parece celular. Espera-se que esses dados possam contribuir para o entendimento da xilogênese e de como os genes trabalham para gerar árvores com características tão distintas, podendo direcionar o melhoramento das mesmas / Abstract: Cellulose is the most abundant polymer in the world and it is present mainly in secondary cell walls of plant mature cells. The knowledge of molecular mechanisms involved in biosynthesis and regulation of this process is recent despite its great importance. The Eucalyptus is the most planted forest genus in the world, especially for being raw material for pulp production. Investments in research and development, especially in the molecular biology field, are being carried out by the forestry sector in order to increase pulp productivity. The data analyzed showed that gene expression of the species the tree species studied here (E. globulus, E. grandis e E. urophylla) differs in essential genes of cell wall compounds formation. Molecular data are consistent with differential data found here in relation to the composition of cell wall sugars. The results generated by the wall composition analysis by various techniques, including the recently developed "Glycome Profiling", showed a wide diversity of carbohydrate of cell wall differently distributed in the xylem of the the Eucalyptus species. These results are a breakthrough in understanding the cell wall composition of these species. The objective of this study was to correlate molecular data with data highthroughput Glycome Profiling to understand the composition and architecture of the cell walls. It is hoped that these data will contribute to the understanding of wood formation and how genes work to generate trees with such different characteristics / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

This dissertation focuses on the development of analytical methods based on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and their applications for separation and structural characterization of oligosaccharides. Porous graphitized carbon liquid chromatography (PGC-LC), gated-trapped ion mobility spectrometry (Gated-TIMS), and electronic excitation dissociation tandem mass spectrometry (EED MS/MS) are three essential techniques employed here. First, the EED method was optimized to generate more informative glycan tandem mass spectra for accurate structural analysis. Glycans were reduced and permethylated or labeled with a reducing-end fixed charge to increase sensitivity, avoid gas-phase structural rearrangement, and facilitate spectral interpretation. EED of glycans produced nearly complete series of Z-, Y- and 1,5X-ions, that appear in the spectra as triplets with characteristic spacing, thus facilitating accurate determination of the glycan topology. Additional radical-driven dissociation pathways were identified, from which different types of linkage-diagnostic ions (cross-ring, secondary, or internal fragments) were generated. The results demonstrated that linkage analysis can be accomplished by utilizing one or a combination of several linkage-diagnostic fragments. EED MS/MS was then implemented, in conjunction with PGC-LC or Gated-TIMS, for on-line separation and characterization of complex mixtures of glycans. These two methods were successfully applied for high-throughput and detailed structural analysis of N-glycans released from human serum, O-glycans released from bovine submaxillary mucin and free oligosaccharides. The performance of these methods was tested and improved through analysis of different types of glycans from a variety of biological sources. Finally, in collaboration with bioinformaticians, a spectral interpretation algorithm, GlycoDeNovo, has been developed for automated and de novo glycan topology reconstruction from their tandem mass spectra. A large number of EED tandem spectra of glycan standards generated in house were used as the training dataset to establish appropriate IonClassifiers for candidate ranking. GlycoDeNovo is capable of identifying correct topologies from MS/MS spectra of glycans in different derivatized forms. Several aspects of this collaborative project were covered in this thesis, including glycan derivatization, data acquisition and manual spectral interpretation to guide the development and evaluate the performance of the automated approach. In this thesis research, integrated approaches utilizing PGC-LC–EED-MS/MS and Gated-TIMS–EED-MS/MS, and the appropriate bioinformatics software, have been established for structural analysis of glycan mixtures. They hold great potential for comprehensive, automated, and de novo glycome characterization.

No description available.

Kam, Kin Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 169-192). / Abstracts in English and Chinese. / Chapter 1. --- Abstract --- p.ii / English --- p.ii / Chinese --- p.v / Chapter 2. --- Acknowledgments --- p.vii / Chapter 3. --- Abbreviations and N-glycan representation --- p.viii / Chapter 4. --- Introduction --- p.1 / Chapter 5. --- Review of Literatures --- p.2 / Chapter 5.1. --- Introduction to Liver Fibrosis --- p.2 / Chapter 5.1.1. --- Pathogenesis of Liver Fibrosis --- p.2 / Chapter 5.1.2. --- Changes of liver architecture - basis of liver fibrosis diagnosis --- p.4 / Chapter 5.2. --- Current Diagnosis of Liver Fibrosis - from Biopsy Examination to Serum Test --- p.5 / Chapter 5.3. --- Glycomics and its Potential as Biomarkers --- p.9 / Chapter 5.3.1. --- Overview of Biochemical and Functional Characteristics of Glycan --- p.13 / Chapter 5.3.2. --- N-linked and O-linked Glycosylations - A Valuable Source of Biomarkers --- p.15 / Chapter 5.3.3. --- Glycomics 一 An Uprising Approach for Biomarker Discovery --- p.17 / Chapter 5.3.4. --- Human Proteome Organisation Human Disease Glycomics/Proteome Initiative --- p.19 / Chapter 5.3.5. --- Recent Applications of Glycomics to Biomarker Discovery --- p.20 / Chapter 5.4. --- Current Technologies for Glycomic Study --- p.22 / Chapter 5.4.1. --- MALDI-TOF MS --- p.22 / Chapter 5.4.2. --- Lectin Microarray --- p.25 / Chapter 5.4.3. --- Liquid Chromatography --- p.27 / Chapter 5.4.4. --- Capillary Electrophoresis --- p.29 / Chapter 5.4.5. --- Quantitative Profiling of Tissue Glycome --- p.31 / Chapter 6 --- Project Rationales and Objectives --- p.36 / Chapter 7 --- Section 1: Methodology Development of Quantitative N- glycomic Profiling --- p.37 / Chapter 1. --- Introduction --- p.37 / Chapter 2. --- Method and Materials --- p.39 / Chapter 3. --- Results --- p.46 / Chapter 4. --- Discussion --- p.65 / Chapter 5. --- Conclusion --- p.71 / Chapter 8. --- Section 2: Serum N-glycomic Profile as Biomarker for Liver Fibrosis 一 Pilot Study --- p.73 / Chapter 1. --- Introduction --- p.73 / Chapter 2. --- Method and Materials --- p.75 / Chapter 3. --- Results --- p.79 / Chapter 4. --- Discussion --- p.86 / Chapter 5. --- Conclusion --- p.94 / Chapter 9. --- Section 3: Serum N-glycomic Profile as Biomarker for Liver Fibrosis -Verification Study --- p.96 / Chapter 1. --- Introduction --- p.96 / Chapter 2. --- Method and Materials --- p.98 / Chapter 3. --- Results --- p.104 / Chapter 4. --- Discussion --- p.137 / Chapter 5. --- Conclusion --- p.152 / Chapter 10. --- General Discussion --- p.153 / Chapter 11. --- Conclusion --- p.167 / Chapter 12. --- Original Data --- p.168 / Chapter 13. --- References --- p.169 / Chapter 14. --- Publications --- p.196

Malaria caused by the protozan parasite Plasmodium is one of the most serious infectious diseases in the developing world. It is estimated that malaria causes an annual mortality rate of ~627,000. New drugs are urgently required, as the incidence of resistance is spreading rapidly. Glycosylphosphatidylinositol (GPI) anchored proteins decorate the merozoite surface and several of which, including merozoite surface proteins - 1 and -2 have previously been shown to be essential for erythrocyte invasion and parasite survival. Plasmodium GPI-anchors contain a glycan core consisting of four mannose residues. Therefore, the enzymes involved in the synthesis of activated mannose, guanidine diphosphomannose pyrophosphorylase (GDP-Man PP) and dolichol phosphate mannose synthase (DPMS), are thought to be crucial for GPI-anchor biosynthesis and as such potential drug targets. Double homologous recombination has been exploited to test whether PfGDP-Man PP and PfDPMS are essential during the erythrocytic portion of the parasite's life cycle. Additionally, overexpression parasite lines for both enzymes have been generated and have shown that the regulation of the two enzymes are intricately linked. Focused metabolomics by multi-reaction monitoring of the overexpression lines suggests that the fucosylation pathway may have a novel function within the parasite, possibly as a dynamic store for activated fucose/mannose. In order to determine the cellular concentration of key metabolites within the parasite, the volumes of the intra-erythrocytic stages have been determined and show that the concentration of metabolites in the ring stage parasites is substantially higher than previously thought. Furthermore, the sub-cellular localisation of GDP-Man PP and DPMS has been determined by immunofluorescence assay. The recombinant expression of DPMS in E. coli allowed its active site residues to be probed as well as establishing a platform for inhibitors to be screened against the enzyme. Finally, inhibitors of the T. brucei DPMS enzyme have been screened against the P. falciparum parasites in culture.