Abstract
The immune system is a complex system of molecules, cells, tissues and organs that protects higher organisms from pathogens or cancerous cells. A proper understanding of the immune system is vital for the development of new vaccines against infectious diseases or immunotherapies against cancer. Mass Spectrometry-based proteomics is an important
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analytical strategy in vaccinology that enables identification and quantification of proteins in cells or tissue, under normal or treated (diseased) conditions. This thesis covers major methodological aspects in the proteomics workflow, from sample enrichment to peptide identification. Applied to different research questions these methods address challenges in sample complexity and sensitivity, as difficulties in peptide sequencing. A positional proteomics strategy was introduced for the selective isolation of N-terminal peptides. This method effectively reduces the complexity of proteomes and enables the global characterization of N-terminal post translation modifications. The positional proteomics strategy was applied to quantitatively profile the protein content of Neisseria Meningitidisouter membrane vesicles (OMV)-based vaccines. Differences in protein content were found between OMV vaccines produced under different purification conditions. The quantitative proteomics data were sustained by serum blot proteomics, confirming a differential pattern in the antigenic protein content. This thesis furthermore describes the development of a two dimensional (2D) chromatographic separation technique for highly sensitive analysis of complex peptide mixtures. The method employs ion exchange chromatography and reversed phase chromatography in an online configuration. The introduction of a novel buffer system omitted the use of undesirable salts, resulting in an excellent compatibility between 2D liquid chromatography and mass spectrometry. Another part of the thesis describes the utility of a recently introduced fragmentation method for the identification of Human Leukocyte Antigens (HLA) class I-associated peptides. This fragmentation technique employs dual fragmentation to generate both the fragment ions induced by Electron-Transfer Dissociation (ETD) and Higher-energy collision Dissociation (HCD) in a single spectrum. The identification of endogenous peptides considerably benefits from the complementary and informative-rich fragmentation spectra provided by this approach. Novel features in the antigen processing machinery are reported, including a variety of post translational modifications for which evidence is accumulating that they play important roles in human diseases.
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