title: Large-scale assignment of N-glycosylation sites using complementary enzymatic deglycosylation.
abstract: 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.
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
mesh_terms: D000818:Animals; D001665:Binding Sites; D002853:Chromatography, Liquid; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D008099:Liver / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D013058:Mass Spectrometry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D040901:Proteomics / Q000379:methods*; D051381:Rats
chemical_list: D006023:Glycoproteins; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase