UC San Diego

Organisms exhibit a vast catalog of mechanisms to target, capture, and disable foreign agents that have infiltrated the body. One major driver of immunogenic response to infection is the production, interaction, and regulation of proteins at the blood-tissue interface. These proteins have several key features that enable them to combat harmful microorganisms. Constituent factors of host proteins such as composition, 3D structure, post-translational modifications, and interaction partners allow for both innate nonspecific response and targeted adaptive response in the fight against pathogens. Mass spectrometry, the analytical technique to measure the mass-to-charge ratio of ions, has long been used for the identification, characterization, and quantification of proteins in vivo and ex vivo. Recent advances have taken advantage of this straightforward experimental design to measure complex post-translational modifications of proteins, including but not limited to glycosylation. Here I have chosen to focus on a major population of the blood-tissue interface during infection, the glycoproteins and their interaction partners. In the first chapter, I describe our large-scale mass spectrometry based proteomic studies of the blood-tissue interface in murine models of infection. In these studies, I have characterized the largest-to-date time-resolved proteomic atlas of the blood-tissue interface during methicillin-resistant Staphylococcus aureus bacteremia. I introduce the idea that major blood and vascular remodeling during infection is organ-specific, highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. Additionally, this chapter highlights the importance of the blood-tissue interface during infectious disease and displays many proteomic and glycoproteomic changes which represent vascular degradation, subsequent organ failure, and death. In the second chapter, I describe mechanisms by which the blood-tissue interface is impacted by pathogens exploiting proteolytic and glycan degradation of immunoglobulins. Using in vivo murine models of group A streptococcal infection, we showcase that human immunoglobulins and a variant of mouse immunoglobulin is specifically degraded after bacterial dissemination to organs. These results stimulate concern for the evaluation of antibody-based therapies in current animal models of streptococcal infections. Finally, I introduce the relationship of glycosylation and antibody function during immunogenic response to infection and highlight the importance of immunoglobulin G which will be a major topic of the third chapter. In the third chapter, I introduce our novel pipeline for identification and quantification of glycopeptides using MS1 & MS2 levels of mass spectrometry data. We have chosen to expand our quantification methods of the glycoproteome to gain greater insight of the heterogeneity of the blood-tissue interface during infection. Here we have leveraged several advancements in the analysis of mass spectrometry data to expand the scope of glycopeptide identification and strengthen the quantification of these biological units. I will describe how I have built a computational pipeline which links the discovery of new glycopeptides from large traditional mass spectrometry datasets to identification and quantification of these glycopeptides in data independent acquisition (DIA) mass spectrometry experiments. Additionally, I analyze the heterogeneity found in the glycosylation of circulating proteins in the blood-tissue interface in models of infection and cell systems used for recombinant antibody production.