| Title | Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry |
| Authors | Zhang, Yong Xie, Xinfang Zhao, Xinyuan Tian, Fang Lv, Jicheng Ying, Wantao Qian, Xiaohong |
| Affiliation | Beijing Inst Radiat Med, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing 102206, Peoples R China. Peking Univ, Renal Div, Hosp 1, Inst Nephrol,Key Lab Renal Dis,Minist Hlth China, Beijing, Peoples R China. Peking Univ, Minist Educ, Key Lab Chron Kidney Dis Prevent & Treatment, Beijing, Peoples R China. Beijing Inst Radiat Med, Beijing 100850, Peoples R China. |
| Keywords | Human serum O-Glycoproteome Site-specific O-glycosylation EThcD COLLISION DISSOCIATION ETHCD CRESCENTIC IGA NEPHROPATHY HUMAN CEREBROSPINAL-FLUID SOLID-PHASE EXTRACTION PROTEIN GLYCOSYLATION ELECTRON-TRANSFER GLYCOPEPTIDE-ENRICHMENT PLASMA PROTEINS INHIBITOR DISEASE |
| Issue Date | 2018 |
| Publisher | JOURNAL OF PROTEOMICS |
| Citation | JOURNAL OF PROTEOMICS. 2018, 170, 14-27. |
| Abstract | Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macro heterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) 0-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. Biological significance: The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took advantage of the inherent properties of the new fragmentation method called EThcD, which provides more complete fragmentation information about O-glycosylated peptides and a more confident site localization of O-glycans than collision-induced dissociation (HCD). We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD was not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Finally, we got a largest data set of site-specific native O-glycoproteome from human serum samples. Furthermore, quantitative analysis of intact O-glycopeptides from the serum samples of IgA nephropathy (IgAN) patients and healthy donors was performed, and the results showed the potential of the strategy to discover O-glycosylation biomarkers. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and lead to exciting opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. |
| URI | http://hdl.handle.net/20.500.11897/500031 |
| ISSN | 1874-3919 |
| DOI | 10.1016/j.jprot.2017.09.014 |
| Indexed | SCI(E) PubMed |
| Appears in Collections: | 第一医院 |
