1. Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative Glycoproteomic characterization.
- Author
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Hua S, Hu CY, Kim BJ, Totten SM, Oh MJ, Yun N, Nwosu CC, Yoo JS, Lebrilla CB, and An HJ
- Subjects
- Amino Acid Sequence, Animals, Bacterial Proteins chemistry, Carbohydrate Conformation, Carbohydrate Sequence, Chromatography, High Pressure Liquid, Glycosylation, Humans, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments isolation & purification, Peptide Mapping, Polysaccharides chemistry, Polysaccharides isolation & purification, Proteolysis, Proteomics, Ribonucleases chemistry, Tandem Mass Spectrometry, Glycoproteins chemistry, Peptide Hydrolases chemistry, Protein Processing, Post-Translational
- Abstract
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.
- Published
- 2013
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