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

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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
*E-mail: [email protected]; phone: 1-613-562-5800, ext. 8674.
Cite this: J. Proteome Res. 2016, 15, 6, 2080–2086
Publication Date (Web):May 5, 2016
https://doi.org/10.1021/acs.jproteome.6b00056
Copyright © 2016 American Chemical Society

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    Abstract

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    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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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00056.

    • Figure S1, evaluating the efficiency of detergent removal off of hydrazide beads by LC-MS/MS analysis of nonglycopeptides released by trypsin digestion; Figure S2, comparing the LC-MS profile (base peak) of nonglycopeptides released by trypsin digestion of fetuin captured on hydrazide beads with or without the presence of detergents; Figure S3, comparing the sequence coverage of fetuin identified from nonglycopeptides by trypsin digestion of fetuin captured on hydrazide beads with or without the presence of detergents; Figure S4, comparing the LC-MS profile (base peak) of glycopeptides released by PNGase F deglycosylation of fetuin captured on hydrazide beads with or without the presence of detergents; Figure S5, comparison-identified deamidated sites identified from the “non-glyco” and “glycol” fractions; Table S1, list of N-glycosites identified as “non-glyco” and “glycol” fractions; Table S2, list of N-glycosites identified from the membrane of HEK 293T cells; Table S3, list of proteins identified from the membrane of HEK 293T cells; Table S4, list of N-glycosites identified from human serum; Table S5, list of proteins identified from human serum (PDF)

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