Abstract
Chapters 2, 3, and 4 describe and demonstrate the intact immunoglobulin protein profiling technique, which was employed to assess the serum of milk antibody repertoire in individual donors. Chapter 2 introduces the method for analysis of the serum IgG1 clonal repertoires of a cohort of sepsis patients. Herein we made
... read more
the striking discoveries that a person’s IgG1 clonal repertoire is rather simple; being dominated by only a few hundreds of clones. The repertoire is unique for each individual and is dynamic in the abundance and presence of detectable clones. Furthermore, Chapter 2 provides proof of concept for the complete de novo sequencing of an abundant endogenous IgG1 clone using a hybrid mass spectrometry approach combining top/middle-down and bottom-up proteomics. Chapter 3 expands the intact antibody profiling method to allow for profiling of IgA1 antibody repertoires by utilization of a protease that specifically cleaves N-terminally of O-glycans, which are specifically found in the hinge regions of IgA1 molecules. We observed that human IgA1 repertoires are relatively simple, unique, and dynamic. Chapter 3 shows that the profiling technique can also be used for the analysis of IgA1 repertoires. Chapter 4 explores the clonal and structural commonalities of IgA1 molecules within individual donors’ serum and milk. It provides proof of shared antibody clones present in both serum and milk of individual donors, even though serum and milk antibodies appear in different oligomeric structures. These oligomeric structures are quantified with mass photometry and after separation of these different oligomeric structures, the data suggests that predominantly the repertoire of dimeric J-coupled IgA in serum harbors clones that are shared with the secretory IgA repertoire in milk. Chapter 5 describes the investigation of the glycosylation of different HLA class I molecules. Glycopeptides originating from the three classical HLA genes A, B, C and the less common HLA F gene were studied. Our data revealed differences in the structure and abundance of the different glycoforms they each harbor. Further investigations suggested that the specific structures and abundances of the glycoforms are likely linked to the subcellular localization of the HLA molecules. In addition, for certain HLA allotypes significant fractions of the molecules may reside intracellularly, whereas for others they are primarily located at the plasma membrane. Finally, Chapter 6 provides a summary and a perspective on how advancements in mass spectrometry instrumentation and methodology have made it possible to perform studies into these earlier considered to be challenging areas of the proteome, and I provide a forward-looking view on where the field may go in the coming years.
show less
