title: Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis.
abstract: Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazide groups to oxidized glycoproteins, thus increasing the coverage of the N-glycoproteome. Compared with the conventional glycopeptide capture method, the detergent-assisted glycoprotein capture approach nearly doubled the number of N-glycosylation sites identified from HEK 293T cells with improved specificity. Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293T cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Furthermore, the application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immunodepletion or fractionation. Overall, the detergent-assisted glycoprotein capture method simplified the capture process, and it increased the number of sites observed on both hydrophobic membrane proteins and hydrophilic secreted proteins.
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa , 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, National Chromatography R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Science , Dalian 116023, China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa , 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D003902:Detergents / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D044967:Serum / Q000737:chemistry
chemical_list: D003902:Detergents; D006023:Glycoproteins; D008565:Membrane Proteins; D020543:Proteome
keywords: N-glycoproteome; detergent; glycoprotein capture; membrane; serum