Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis

Author

Rui Chen^†, Hanfa Zou^‡ and Daniel Figeys^*†

*E-mail: dfigeys@uottawa.ca; phone: 1-613-562-5800, ext. 8674.

†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

‡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

Journal of Proteome Research

Vol. 15: Issue. 6: Pages. 2080-2086

Publication Date (Web): May 5, 2016

DOI: 10.1021/acs.jproteome.6b00056

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.

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http://pubs.acs.org/doi/abs/10.1021/acs.jproteome.6b00205#/doi/full/10.1021/acs.jproteome.6b00264