Large-Scale and Site-Specific Mapping of the Murine Brain O -Glycoproteome with IMPa.

Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available O -glycoprotease IMPa from Pseudomonas aeruginosa for comprehensive O -glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O -glycopeptides were analyzed by electron-transfer/higher-energy collision dissociation (EThcD), which permits site-specific and global analysis of all types of O -glycans. We developed two complementary approaches for the analysis of the total O -glycoproteome using HEK293 cells and derivatives. The results demonstrated that IMPa and EThcD facilitate the confident localization of O -glycans on glycopeptides. We then applied these approaches to characterize the O -glycoproteome of the mouse brain, which revealed the high frequency of various sialylated O -glycans along with the unusual presence of the Tn antigen. Unexpectedly, the results demonstrated that glycoproteins in the brain O -glycoproteome only partly overlap with those reported for the brain N -glycoproteome. These approaches will aid in identifying the novel O -glycoproteomes of different cells and tissues and foster clinical and translational insights into the functions of protein O -glycosylation in the brain and other organs.