Heterogeneity of glycoproteins: Why does it matter and how to account for it

Glycosylation can be critical for determining the structure and functions of proteins, but it is often neglected, leading to significant knowledge gaps in our understanding of biology. The inherent heterogeneity of glycans presents technical challenges to glycoprotein characterisation and impedes the representation of intact glycoprotein structures. Here we discuss how glycan heterogeneity constitutes a fundamental property of glycoproteins and acts as a remarkably powerful strategy for modulating biological function on the fly, complementing the rigidity of the genome. We present recent examples of how integrating mass spectrometry glycoproteomics and glycomics, structural biology, and molecular dynamics simulation data can bring glycans into a 3D structural framework, providing a unique perspective into the roles of glycosylation and potentially accelerating the design of glycoprotein biologics. Strengths and limitations of these approaches are also highlighted.