Large-scale assignment of N-glycosylation sites using complementary enzymatic deglycosylation

Endoglycosidase is a class of glycosidases that specifically cleaves the glycosidic bond between two proximal residues of GlcNAc in the pentasaccharide core of N-glycan, leaving the innermost GlcNAc still attached to its parent protein, which provides a different diagnostic maker for N-glycosylation site assignment. This study aims to validate the use of endoglycosidase for high throughput N-glycosylation analysis. An endoglycosidase of Endo H and the conventional PNGase F were employed, with a similar accessible procedure, for large-scale assignment of N-glycosylation sites and then N-glycoproteome for rat liver tissue. ConA affinity chromatography was used to enrich selectively high-mannose and hybrid glycopeptides before enzymatic deglycosylation. As a result, a total of 1063 unique N-glycosites were identified by nano liquid chromatography tandem mass spectrometry, of which 53.0% were unknown in the Swiss-Prot database and 47.1% could be assigned only by either of the methods, confirmed the possibility of large-scale glycoproteomics by use of endoglycosidase. In addition, 11 glycosites were assigned with core-fucosylation by Endo H. A comparison between the two enzymatic deglycosylation methods was also investigated. Briefly, Endo H provides a more confident assignment but a smaller dataset compared with PNGase F, showing the complementary nature of the two N-glycosite assignment methods.