title: From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins.
abstract: O-glycosylation-site characterization of individual glycoproteins is a major challenge because of the heterogeneity of O-glycan core structures. In proteomic studies, O-glycosylation-site analysis is even more difficult because of the complexity of the sample. In this work, we designed a rapid and convenient workflow for characterizing the O-glycosylation sites of individual proteins and the human-plasma proteome. A mixture of exoglycosidases was used to partially remove O-glycan chains and leave an N-acetylgalacosamine (GalNAc) residue attached to the Ser or Thr residues. The O-glycosylated peptides could then be identified by using liquid chromatography-tandem mass spectrometry (LC-MS-MS) to detect the 203 Da mass increase. Jacalin was used to selectively isolate O-GalNAc glycopeptides before LC-MS-MS analysis, which is optional for individual proteins and necessary for complex human-plasma proteins. Bovine fetuin and human chorionic gonadotropin (hCG) were used to test the analytical workflow. The workflow indicated superior sensitivity by not only covering most previously known O-glycosylation sites but also discovering several novel sites. Using only one drop of blood, a total of 49 O-GalNAc-linked glycopeptides from 36 distinctive glycoproteins in human plasma were identified unambiguously. The approach described herein is simple, sensitive, and global for site analysis of core 1 through core 4 O-glycosylated proteins.
authors: National Glycoengineering Research Center, Shandong University, 250100, Jinan, China.
authors: Xue
authors: X
authors: Bai
authors: Daoyuan
authors: D
authors: Li
authors: Jing
authors: J
authors: Zhu
authors: Yudong
authors: Y
authors: Guan
authors: Qunye
authors: Q
authors: Zhang
authors: Lianli
authors: L
authors: Chi
mesh_terms: D001798:Blood Proteins / Q000737:chemistry*; D006063:Chorionic Gonadotropin / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D020543:Proteome
chemical_list: D001798:Blood Proteins; D006063:Chorionic Gonadotropin; D020543:Proteome