Spatial proteome mapping of specialized subcellular structures in human cells

Proteins are the primary workhorses of the cell, carrying out virtually all processes to sustain cellular functioning. From enzymes that catalyze biochemical reactions, to motor proteins that transport large cellular cargo across the cell, protein functions are as diverse as the unique amino acid sequences that compose the proteins. Protein function is largely dependent on the subcellular localization of the protein, as subcellular compartmentalization enables different environments that are suitable for different reactions. Knowledge about protein localization and function can, in the broader context, help us understand the cell in health and disease, as protein dysfunction and mislocalization are key drivers of developing disease. The work in this thesis has been carried out within the framework of the Human Protein Atlas (HPA) initiative, primarily for the subcellular resource. In Paper I, we measured the autoantibody profiles of patients with systemic sclerosis with the goal to identify new candidate biomarkers associated with fibrosis. We performed a near proteome-wide, untargeted screen combined with a targeted bead array and revealed 11 autoantibodies with higher prevalence in patients with systemic sclerosis than in controls. Two of these show high potential for being used as biomarkers for systemic sclerosis patients that are affected by skin and lung fibrosis. For Paper II, we took advantage of the vast image library generated by the subcellular resource of HPA to create an image-based map of the micronuclear proteome. In total, we identified 944 proteins as micronuclear, dominated by proteins associated with nuclear and chromatin processes. The findings of this study expand our view of micronuclei as byproducts of mitotic errors to potential active participants in biological processes. In Paper III, we applied antibody-based spatial proteomics combined with 3D confocal imaging to map 715 proteins to primary cilia, and three ciliary substructures, across three different cell lines. Of the identified proteins, 91 had not been identified in cilia before, expanding our knowledge on the ciliary proteome and function. The findings of the study portray cilia as sensors able to tune their proteome to effectively sense the environment to compute cellular responses. Finally, in Paper IV, we mapped the subcellular localization of a subset of the human sperm proteome to 11 distinct subcellular structures of human sperm cells, providing the first image-based resource on protein localization in sperm cells. We found that 54% of the studied sperm proteins vary in spatial distribution and/or abundance between individual sperm, which raises the question of subpopulations of sperm. In summary, this thesis expands our knowledge on protein localization in specialized subcellular structures and provides a foundation for further in-depth research into the mechanisms behind the drivers of certain diseases, such as for autoimmunity, cancers, ciliopathies, and male infertility phenotypes.