Deciphering the Functions of O-GlcNAc Glycosylation in the Brain: The Role of Site-Specific Quantitative O-GlcNAcomics

The dynamic posttranslational modification O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) is present on thousands of intracellular proteins in the brain. Like phosphorylation, O-GlcNAcylation is inducible and plays important functional roles in both physiology and disease. Recent advances in mass spectrometry (MS) and bioconjugation methods are now enabling the mapping of O-GlcNAcylation events to individual sites in proteins. However, our understanding of which glycosylation events are necessary for regulating protein function and controlling specific processes, phenotypes, or diseases remains in its infancy. Given the sheer number of O-GlcNAc sites, methods for identifying promising sites and prioritizing them for time- and resource-intensive functional studies are greatly needed. Revealing sites that are dynamically altered by different stimuli or disease states will likely go a long way in this regard. Here, we describe advanced methods for identifying O-GlcNAc sites on individual proteins and across the proteome and for determining their stoichiometry in vivo. We also highlight emerging technologies for quantitative, site-specific MS-based O-GlcNAc proteomics (O-GlcNAcomics), which allow proteome-wide tracking of O-GlcNAcylation dynamics at individual sites. These cutting-edge technologies are beginning to bridge the gap between the high-throughput cataloguing of O-GlcNAcylated proteins and the relatively low-throughput study of individual proteins. By uncovering the O-GlcNAcylation events that change in specific physiological and disease contexts, these new approaches are providing key insights into the regulatory functions of O-GlcNAc in the brain, including their roles in neuroprotection, neuronal signaling, learning and memory, and neurodegenerative diseases.