Glyco-analytical multispecific proteolysis (Glyco-AMP): A simple method for detailed and quantitative glycoproteomic characterization

  • Hua S
  • Hu C
  • Kim B
 et al. 
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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.

Author-supplied keywords

  • biopharmaceutical glycoproteins
  • glycan isomers
  • glycoproteomics
  • multispecific proteases
  • nonspecific proteases
  • site-specific glycosylation

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Authors

  • Serenus Hua

  • Chloe Y. Hu

  • Bum Jin Kim

  • Sarah M. Totten

  • Myung Jin Oh

  • Nayoung Yun

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