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J Proteome Res. 2013 Oct 4;12(10):4414-23. doi: 10.1021/pr400442y. Epub 2013 Sep 20.

Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative Glycoproteomic characterization.

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1
Asia Glycomics Reference Site , Daejeon 305-764, South Korea.

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.

PMID:
24016182
DOI:
10.1021/pr400442y
[Indexed for MEDLINE]
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