MHC class I-associated peptides are presented at the cell surface for scrutiny by CD8+ T lymphocytes and are collectively referred to as the immunopeptidome. The immunopeptidome plays crucial roles in many processes including autoimmunity, cancer immunosurveillance and elimination of pathogen-infected cells. Nevertheless, very little is known about the molecular composition and biogenesis of the immunopeptidome in health and disease. Through a multidisciplinary effort, I will conduct both fundamental and applied immunopeptidomic studies by using cutting-edge technologies in mass spectrometry(MS)-based proteomics. First, I will apply systems-level approaches to investigate the global relationship between the immunopeptidome, the proteome and the degradome in human cells under normal physiological conditions, i.e. in the absence of infection. This will allow us to gain new fundamental insights into the biogenesis of the immunopeptidome and will provide general concepts of how to manipulate MHC I peptide processing and presentation in immunity. Second, applied research will be conducted by mining the immunopeptidome of human cells infected by the life-threatening pathogen Mycobacterium tuberculosis (Mtb). Each year, almost two million people die of tuberculosis (TB) raising the urgent need of a new and more efficient vaccine against TB. In this project, we aim to discover numerous MHC I peptides encoded by Mtb proteins that could be further evaluated as vaccine candidates against TB. Altogether, the impact of this project on EU competitiveness will be enormous by accelerating the rational design of immunotherapeutic interventions against autoimmunity, cancers and infectious diseases, TB in particular.