Proteomics and biotechnologies: new methods for glycoproteome analysis
Giangrande, Chiara (2011) Proteomics and biotechnologies: new methods for glycoproteome analysis. [Tesi di dottorato] (Inedito)
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Biological systems are made up of a plethora of organic components. Since nowadays attention has been focused on two important classes of bioinformative molecules, nucleic acids and proteins, whose large scale study has led to the rise of the so called “omics” sciences, genomics and proteomics. In particular the study of the proteome implies not only the protein complement of a given cell, but even the study on a high throughput scale of proteins post translational modifications, interactions, and functions. More than 50% of mammalian proteins are glycosylated and this observation has led to the conclusion that sugars attachment broadens variability among gene products. Glycans generally cover cellular surfaces, ranging from viruses to the most complex multicellular organisms and they can be considered as a molecular code that dictates to cells how to communicate with each other. The wide range of important biological processes mediated by carbohydrates has given origin to glycomics, the large scale study of the whole set of glycans of an organism. This PhD thesis targeted the development of methodological platforms for the study of glycoproteins and glycoconjugates by the integration of affinity chromatography strategies together with high performance liquid chromatography and mass spectrometry. My first steps in the study of glycosylation were focused on the development of enrichment, derivatization, and mass spectrometry procedures, that were applied to the study of the protein content of egg. Peptides of egg glycoproteins, bearing N-glycosylation sites, were captured by Concanavalin A affinity chromatography and detected by LC-MS/MS after deglycosylation. Oligosaccharides were analyzed by MALDI-MS/MS before and after dansylhydrazine derivatization of the reducing end. This derivatization was introduced to enhance oligosaccharides fragmentation characteristics. Attempts to achieve more structural information on glycans were carried out using multi-stage mass spectrometry on MALDI-LTQ-Orbitrap. A proteomic approach was employed to investigate the molecular bases of myocarditis, a group of diseases that have in common the inflammation of the heart. In this case, the study of glycosylation anomalies in the sera sample of myocarditis-affected patients came after a wider study including both the analysis of free peptides and a general overview on protein content by 2D gel electrophoresis. This study represents a starting point for further studies aiming at the screening of proteic biomarkers for this pathological status and involving different fields of clinical investigations, opening up new opportunities for therapeutics and early diagnostics. Glycoproteins-based therapeutics are generally produced in mammalian host systems, but plants can be considered a valuable alternative for their ability to produce homogeneously glycosylated recombinant proteins. The possibility of exploiting plants as host systems and the lack of consistent information concerning glycosylation in the plant model system Arabidopsis thaliana encouraged me to investigate its glycoproteome, using the strategies previously developed. The last part of my work was devoted to the study of innate immunity mechanisms triggered by lipopolysaccharides, bacterial glycoconjugates located on cell surfaces. The full exploitation of glycoconjugates potential for industrial biotechnological and pharmaceutical applications requires a deep knowledge of the immunological mechanisms at the base of host-pathogen recognition. The goal of the project was the development of a strategy to capture LPS-interacting proteins and glycoproteins in human serum, considering that some of these mechanisms are still unknown in non-human biological systems.
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