Cheng Ma1, Ding Liu1, Dong Li2, Junping Zhang3, Xiao-Qian Xu4, He Zhu1, Xiu-Feng Wan5,6,7,8,9,10, Carol H Miao11,12, Barbara A Konkle12,13, Philip Onigman14, Weidong Xiao3, Lei Li1. 1. Department of Chemistry, Georgia State University, Atlanta, GA, USA. 2. Department of Clinical Laboratory, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China. 3. Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Cardiovascular Research Center, Temple University, Philadelphia, PA, USA. 4. Department of Hematology, Shanghai Jiaotong University Affiliated Shanghai General Hospital, Shanghai, China. 5. Missouri University Center for Research on Influenza Systems Biology (CRISB), University of Missouri, Columbia, MO, USA. 6. Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USA. 7. Department of Electrical Engineering & Computer Science, College of Engineering, University of Missouri, Columbia, MO, USA. 8. Bond Life Sciences Center, University of Missouri, Columbia, MO, USA. 9. MU Informatics Institute, University of Missouri, Columbia, MO, USA. 10. Department of Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA. 11. Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, USA. 12. University of Washington, Seattle, WA, USA. 13. Bloodworks Northwest, Seattle, WA, USA. 14. Genovis Inc, Cambridge, MA, USA.
Abstract
BACKGROUND/ OBJECTIVE: Coagulation factor V (FV), a multidomain glycoprotein, is an essential cofactor in the blood clotting cascade. FV deficiency is a rare bleeding disorder that results in poor clotting after an injury or surgery. The only treatment for the disease is infusions of fresh frozen plasma and blood platelets. Glycosylation affects the biological activity, pharmacokinetics, immunogenicity, and in vivo clearance rate of proteins in the plasma. The glycan profile of FV, as well as how it affects the activity, stability, and immunogenicity, remains unknown. METHODS: In this study, we comprehensively mapped the glycosylation patterns of human plasma-derived FV by combining multienzyme digestion, hydrophilic interaction chromatography enrichment of glycopeptides, and alternated fragmentation mass spectrometry analysis. RESULTS/ CONCLUSION: A total of 57 unique N-glycopeptides and 51 O-glycopeptides were identified, which were categorized into 40 N-glycan and 17 O-glycan compositions. Such glycosylation details are fundamental for future functional studies and therapeutics development. In addition, the established methodology can be readily applied to analyze glycosylation patterns of proteins with more than 2000 amino acids.
BACKGROUND/ OBJECTIVE: Coagulation factor V (FV), a multidomain glycoprotein, is an essential cofactor in the blood clotting cascade. FV deficiency is a rare bleeding disorder that results in poor clotting after an injury or surgery. The only treatment for the disease is infusions of fresh frozen plasma and blood platelets. Glycosylation affects the biological activity, pharmacokinetics, immunogenicity, and in vivo clearance rate of proteins in the plasma. The glycan profile of FV, as well as how it affects the activity, stability, and immunogenicity, remains unknown. METHODS: In this study, we comprehensively mapped the glycosylation patterns of human plasma-derived FV by combining multienzyme digestion, hydrophilic interaction chromatography enrichment of glycopeptides, and alternated fragmentation mass spectrometry analysis. RESULTS/ CONCLUSION: A total of 57 unique N-glycopeptides and 51 O-glycopeptides were identified, which were categorized into 40 N-glycan and 17 O-glycan compositions. Such glycosylation details are fundamental for future functional studies and therapeutics development. In addition, the established methodology can be readily applied to analyze glycosylation patterns of proteins with more than 2000 amino acids.
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