Decoding of O-Linked Glycosylation by Mass Spectrometry

Protein glycosylation (N- and O-linked) plays an important role in many biological processes, including protein structure and function. However, the structural elucidation of glycans, specifically O-linked glycans, remains a major challenge and is often overlooked during protein analysis. Recently, mass spectrometry (MS) has matured as a powerful technology for high-quality analytical characterization of O-linked glycans. This review summarizes the recent developments and insights of MS-based glycomics technologies, with a focus on mucin-type O-glycan analysis. Three main MS-based approaches are outlined: O-glycan profiling (structural analysis of released O-glycan), a bottom-up approach (analysis of an O-glycan covalently. attached to a glycopeptide), and a top-down approach (analysis of a glycan attached to an intact glycoprotein). In addition, the most widely used MS ionization techniques, i.e., matrix-assisted laser desorption ionization and electrospray ionization, as well as ion activation techniques like collision-induced dissociation, electron capture dissociation, and electron transfer dissociation Intact Glycoprolehl Glycoprotein MS technical approaches mentioned above are already major improvements for studying O-linked glycosylation and appear to be valuable for in-depth analysis of the type of O-glycan attached, branching patterns, and the occupancy of O-glycosylation sites.