Novel methodologies in quantitative proteomics

The proteome is defined as the set of all expressed proteins in a cell, tissue or organism. Proteome analysis presents specialized analytical problems in two major areas: i) dynamic expression range and ii) diversity of protein expression (multiple protein forms). Post-translational modification of proteins is part of what makes proteomics so much challenging. It is an event with dramatic effects on the complexity of the proteome. Nowadays the attention towards PTMs is notably increased due to the fact that they play a critical role in cellular functioning and they vary in response of environmental stimuli. There are many classical techniques designed to analyse PTMs. This PhD thesis focuses on the development of new proteomics approaches for qualitative and quantitative analysis of protein nitration, protein carbonylation and protein phosphorylation, three of the most important known PTMs. The approaches are mainly based on selective chemical modification of residues of interest coupled to advanced mass spectrometry methods. These approaches aim at a comprehensive analysing of such modifications by identification of the modified proteins, exact localization of the modified residues and quantification of the modification. The first section of this thesis is focused on the development and the optimization of a new strategy for the qualitative and quantitative analysis of nitration of tyrosine residues. Thus, improved approach, based on chemical labelling and mass spectrometric procedures, for the selective analysis of 3-nitrotyrosine residues in protein mixtures is described. However the major aim of this work is devoted to the use of isotopically labelled molecule to allow a simple and direct quantitative MS analysis. The second part aims to the optimization of a labelling protocol of carbonylated residues coupled to advanced mass spectrometry based scanning techniques to improve detection of such residues. Moreover a method for selective quantitative analysis is also described, aimed at the direct quantification of oxidation levels of proteins in different samples. The last part of the work is focused on the development of a strategy for qualitative and quantitative analysis of serine and threonine phosphorylation without preliminary enrichment but using chemical labelling in combination with the high performance of a novel hybrid mass spectrometer Linear Ion Trap. To prove their wide enforceability these methodologies were set up first on model proteins and then applied to more complex systems.