Integrated strategies to develop post-translationally modified proteins in extracellular vesicles as candidate disease markers

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing proteins and nucleic acid cargo. These vesicles are released by almost all cell types and provide an effective and ubiquitous path for intercellular communication and transmission of pathogenic and signaling molecules among cells. Research into potential biomarkers isolated from EV has been propelled by the development of methods and tools to acquire them by minimally and non-invasive means, which reinforces their great diagnostic potential. In the context of cancer, this opens the door to apply EV based liquid biopsy for early detection prior to alternate, more prevailing diagnostic tools like imaging studies. In autoimmune diseases, EVs play a crucial role in immune responses and as immunomodulatory agents as they can modulate the function of a wide variety of immune cells, especially in antigen-presenting cells (APCs). Several efforts have been made to study EVs and their cargo in numerous disease models, but very few in autoimmunity. Autoimmune diseases are chronic, have been underexplored especially in the omics area, and their diagnosis and treatment rely on traditional therapy. Therefore, there is a need for efficient methods to elucidate biomarkers that could provide additional layers of information for treatment, diagnosis, and prognosis. Additionally, protein post-translational modifications (PTMs), such as phosphorylation, glycosylation, and acetylation, are involved in multiple essential cellular processes and represent an important mechanism of regulation for cellular physiological functions, leading to the development of effective and targeted therapeutics. Discovery and profiling PTMs have established the relevance of PTMs in EVs and associated EV functions and novel applications. This dissertation proposes integrated proteomic strategies to efficiently isolate and analyze EVs in human plasma from different types of pathologies like cancer and autoimmune diseases. The main focus is the development of the platforms, to not only isolate the proteome from EVs, but also PTMs including phosphorylation, glycosylation and acetylation, simultaneously. Chapter one, which is the core of this dissertation, describes the platform to sequentially isolate and analyze the EV proteome, phosphoproteome and glycoproteome from human plasma. Chapters two and three focus on the ongoing application of this platform with slight modifications into different disease models, in this case breast cancer subtypes and autoimmune diseases.