Understanding the nuances of intercellular communication with sensitive mass spectrometric approaches

In this thesis, I applied high-sensitivity mass spectrometry (MS) approaches to characterize extracellular vesicles (EVs) and peptide hormones, two key systems for cell-to-cell communication. With thoughtful sample pretreatment and LC-MS/MS strategies, I attempt to overcome technical hurdles to obtain novel biological insights into both systems. This work will lay strong foundation to better understand elements of cell-cell communication for therapy. In Chapter 2, I optimized an immunopeptidomics workflow to work with highly limited amounts of starting material, pushing the limit in the EV and the antigen discovery fields. I expanded the measurable EV antigen repertoire by 8-fold, to ~3,500 HLA peptides. This new depth of identification allowed me to contrast the properties of the EV ligand repertoire to that of the cell surface, revealing that EVs are densely loaded with HLA molecules and over-represent HLA-B and cysteinylated ligands. In Chapter 3, I attempted to study surface features of EVs. Prompted by the lack of structural information of proteins that reside in EV membrane, I pursued structural characterization of protein complexes in EVs, to understand the unique interactions that may enable EV functionality. This led to an intact EV crosslinking (iEVXL) approach, combining crosslinking and MS analysis. With this strategy, we add a novel approach to the .limited toolbox for structural characterization of EVs. In Chapter 4, I applied (phospho)proteomics to characterize the effects that short-term EV-treatment has on cardiomyocyte function, aiming to understand how EVs may support cardiac regeneration. The content of EVs derived from cardiac progenitor cells (CPCs) was examined by MS, which shortlisted PAPPA and NID-1 as potential mediators of endothelial cell activation and migration. In parallel, changes on the phospho-proteome of endothelial cells upon treatment with CPC-EVs revealed altered Insulin/IGF pathway. As PAPPA has been proposed to mediate extracellular release of IFG-1 by disrupting the IGF1/IGFBP4 complex, our data indicates that PAPPA contributes to pro-regenerative properties of CPC-EVs. In Chapter 5 and 6, I applied sensitive MS approaches to study peptide hormone secretion and processing using a panel of human EEC-enrichable organoid models. In Chapter 5, we present an EEC atlas, where we characterized the proteins, receptors, peptidases, and secreted hormones present in various EECs, generating a rich resource to study human EEC development and function. In this atlas we also provide the first transcriptomic and proteomic map of Motilin-producing cells, a human EEC population that cannot be replicated in mice. In Chapter 6, we knocked-out/mutated key enzymes involved in the processing of these hormones inside EECs, and used MS to monitor hormone peptide processing specificity. We identified PCSK2-dependent glucagon production in intestinal EECs, stimulated upon BMP signaling. We also map the substrates and products of major EECs endo/exopeptidases (PCSK1, PCSK2, DPP4, CPE and CPB1), identifying many bioactive peptide hormones, and describing the roles of specific proteases in their generation. Finally, in Chapter 7, I discuss remaining challenges on the field of high-sensitivity MS analysis applied to secreted mediators of cell-to-cell communication, together with the future perspectives for general high-sensitivity MS analysis of peptides and proteins