Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis

Jana Krenkova, Akos Szekrenyes, Zsolt Keresztessy, Frantisek Foret, Andras Guttman

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors.

Original languageEnglish
Pages (from-to)54-61
Number of pages8
JournalJournal of Chromatography A
Volume1322
DOIs
Publication statusPublished - Dec 27 2013

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Keywords

  • Deglycosylation
  • Enzyme microreactor
  • Monolith
  • Oriented immobilization
  • PNGase F

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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