Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis
| Authors | |
|---|---|
| Year of publication | 2013 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Chromatography A |
| MU Faculty or unit | |
| Citation | |
| Doi | http://dx.doi.org/10.1016/j.chroma.2013.10.087 |
| Field | Analytic chemistry |
| Keywords | Enzyme microreactor; Oriented immobilization; Monolith; PNGase F; Deglycosylation |
| Description | 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 succinimidy1-6-(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. (C) 2013 Elsevier B.V All rights reserved. |
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