title: A precise approach in large scale core-fucosylated glycoprotein identification with low- and high-normalized collision energy.
abstract: The core fucosylation (CF) of N-glycoproteins plays important roles in regulating protein functions during biological development, and it has also been shown to be up-regulated in several high metastasis cancer cell lines. Therefore, global profiling and quantitative characterization of CF-glycoproteins may reveal potent biomarkers for clinical applications. However, due to the complex fragmentation pattern of CF-glycopeptides, accurately identifying CF-glycosylation sites via mass spectrometry with high throughput remains a formidable challenge. In this study, we established a precise CF-glycosylation site identification strategy with UHPLC LTQ-Orbitrap Elite under low- and high-normalized collision energy (LHNCE) conditions. To demonstrate the feasibility of LHNCE, the CF-glycopeptides of target proteins in clinical plasma samples were applied and compared as a preliminary demonstration and resulted in the assignment of 357 unique CF-glycosylation sites from 209 CF-glycoproteins. In this study, the largest human plasma CF-glycosylation site database was constructed, and at least three-fold more CF-sites were identified compared to previously published studies. The results further demonstrated that LHNCE provides an important approach for CF-glycoprotein function studies and biomarker screening in cancer research.
  
  
  CONCLUSIONS
  Core-fucosylation (CF) is a kind of N-linked glycosylation in which an α1,6-linked fucose is added to the innermost N-acetylglucosamine (GlcNAc) residue. It has been proved that core-fucosylation is involved in regulating biological processes in mammals. Abnormal core-fucosylation has been demonstrated in human pathological processes, such as metastasis. For example, the CF-glycosylation of an α-fetoprotein isoform (AFP-L3) was approved as a biomarker of hepatocellular carcinoma (HCC). In addition, GP73 is also a well-known biomarker and its CF-glycosylation level will increase in liver cancer patients. Therefore, it is crucial to develop a strategy for mapping human CF-glycosylation.
authors.affiliation: Center for Diagnostics & Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Cheng
authors.initials: C
authors.lastname: Ma
authors.affiliation: Department of Parasitology, Faculty of Biology, Philipps University Marburg, Marburg, Hessen 35043, Germany.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Center for Diagnostics & Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States; National Glycoengineering Research Center, The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, People's Republic of China.
authors.forename: Jingyao
authors.initials: J
authors.lastname: Qu
authors.affiliation: National Institute of Biological Sciences, Beijing 102206, People's Republic of China.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Center for Diagnostics & Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.affiliation: Center for Diagnostics & Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States; National Glycoengineering Research Center, The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, People's Republic of China.
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Center for Diagnostics & Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States; National Glycoengineering Research Center, The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, People's Republic of China. Electronic address: pwang11@gsu.edu.
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005643:Fucose / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods*
chemical_list: D001798:Blood Proteins; D006023:Glycoproteins; D005643:Fucose
keywords: Core-fucosylation; FASP; HCD; HILIC; Human plasma; NCE