Glycoproteomics with AI-ETD - Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

Protein glycosylation is a highly important, yet a poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity and analytical challenge. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has ultimately limited our abilities to both address the degree of heterogeneity across the glycoproteome and to understand how it contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured at a global level via glycopeptide profiling with activated ion electron transfer dissociation (AI-ETD), enabling characterization of nearly 2,100 N-glycosites (> 7,500 unique N-glycopeptides) from mouse brain tissue. Moreover, we have used this unprecedented scale of glycoproteomic data to develop several new visualizations that will prove useful for analyzing intact glycopeptides in future studies. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein.