The Biological Basis and Analyses of N-Glycan Microheterogeneity

N-glycosylation is an abundant and essential co/post-translational modification that is preserved across all eukaryotes. N-glycans have important functions in protein stability and protein-protein interactions. N-glycans exhibit a high degree of heterogeneity, even within an individual site on the same protein, a phenomenon that is termed "microheterogeneity," which is the focus of this review. Traditional analytical approaches with released glycans are limited in their usefulness in studying microheterogeneity because of most glycoproteins having more than one site of N-glycosylation. Since specific N-glycans at specific sites can confer important functions to glycoproteins, this presents a significant gap between the information content of glycomics and glycoproteomics experiments. More recently, tandem mass spectrometry of intact glycopeptides has been used to obtain site-specific information on N-glycan microheterogeneity. The microheterogeneity of glycoproteins presents a significant analytical challenge not only during mass spectrometry analyses but also in downstream data processing. Use of specialized search engines followed by extensive manual validation is often required for accurate and in-depth glycoproteomics. Overall, recent advances in analytical technology and data processing present exciting new opportunities to analyze N-glycans in a site-specific manner. Being able to define, understand functional consequences of, and even predict and direct N-glycan microheterogeneity has implications across many fields, including the manipulation and production of glycoprotein biologics.