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Glyco-Analytical Multispecific Proteolysis (Glyco-AMP): A Simple Method for Detailed and Quantitative Glycoproteomic Characterization

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Asia Glycomics Reference Site, Daejeon 305-764, South Korea
Cancer Research Institute, Chungnam National University, Daejeon 305-764, South Korea
§ Department of Chemistry, University of California—Davis, Davis, California 95616, United States
Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, South Korea
Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang 363-883, South Korea
*Tel: 82 42-821-8547; fax: 82 42-821-8551; e-mail: [email protected] (H.J.A.). Tel: 1 530-752-0504; fax: 1 530-752-8995; e-mail: [email protected] (C.B.L.).
Cite this: J. Proteome Res. 2013, 12, 10, 4414–4423
Publication Date (Web):September 9, 2013
Copyright © 2013 American Chemical Society

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    Abstract Image

    Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.

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