Proteomics and biotechnologies: new methods for glycoproteome analysis

Chiara Giangrande
2011
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,
more » ... nd 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 app [...]
doi:10.6092/unina/fedoa/8586 fatcat:ibt4vj7uq5amzc2fk7htuocvim