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Abstract
N-glycans are essential to eukaryotic biology and are important modulators of glycoprotein folding, stability, binding, and other functions. Glycoproteins often host multiple sites of N-glycosylation, each of which is modified by an extensive non-template process as the glycoprotein folds and travels through the secretory pathway. Each site of N-glycosylation on an individual glycoprotein hosts its own distinct distribution of glycoforms which often denote details of their incomplete processing, a phenomenon termed ‘microheterogeneity’. Due to the importance of N-glycans in the development of biologics and biosimilars, there is great interest in both understanding and controlling N-glycan microheterogeneity to improve the quality control and efficacy of therapeutics. This dissertation looks to understand the underlying causes of microheterogeneity from the perspective of the glycoprotein that is modified and with a focus on the stages of processing that delineate the three major classes of N-glycan: high-mannose, hybrid, and complex. By utilizing modern high-resolution high-accuracy mass spectrometry methods and glycopeptide-focused data analysis, we characterized a set of reporter glycoproteins that host varying numbers and diversities of N-glycans, totaling 38 sites of N-glycosylation. Following the development of this enzyme-specific activity database we looked to identify surrounding structural features that influence N-glycan processing. We identify the importance of protein tertiary structure in defining the relative efficiencies of glycan-processing enzymes towards differing sites. Using protein surface modeling, we identify convexity as a predictor of N-glycan processing efficiency. We also find that proximal phenylalanines may influence the balance between high-mannose and hybrid/complex structures. Additionally, we share a new tool (ppmFixer) for the mass spectrometry search engine pGlyco that dramatically improves the accuracy of its output. Future studies will continue to characterize downstream glycan-processing enzymes as well as the structural determinants that define glycoprotein-glycosyltransferase reactions.