One of the most critical post-translational modifications in proteins is N-glycosylation, influencing the folding and function of ~ 1/3rd of the cellular proteome. N-glycosylation occurs when a preassembled glycan is transferred en bloc to a protein acceptor at specific protein sites (sequons) by the oligosaccharyltransferase. These glycans are then modified as proteins traverse the secretory pathway. Alterations in glycan structure and occupancy at specific sequons have significant clinical and industrial impact, leading to a growing number of diseases (Congenital Disorders of Glycosylation, cancer, etc) and to changes in secretion, half-life, and activity of proteins with biotechnological relevance. Further, glycans influence protein folding, stability, and traffic, and all of these depend on quality control processes that use glycans to decide on a protein’s fate. It is therefore important to understand how glycosylation alterations occur and what is the impact of these changes in mature and immature protein expression. A more complete understanding of the causes and consequences of changes in N-glycosylation will lead to the design of better therapeutics, diagnostic strategies, and industrial bioprocesses.
To more effectively and efficiently study the mechanism of N-glycosylation and its physiological impact throughout the secretory pathway, we combined biochemical subcellular fractionation methods with quantitative SWATH-MS glycoproteomic workflows to measure site-specific and global changes in glycan occupancy and structure. We tested these methods in yeast cells with normal or altered glycan biosynthetic or quality control processes. We expanded these studies to look at the entire proteome and at the quantitative influence of protein quality control on protein maturation. Our protocols allow for rapid relative quantitative and qualitative proteomics and glycoproteomics of a variety of samples. Our methods and results have important implications in the understanding of the fundamentals of the glycosylation and quality control processes and on the industrial and medical applications of glyco-biotechnology.