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

J 2013

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

KRENKOVA, Jana, Akos SZEKRENYES, Zsolt KERESZTESSY, František FORET, Andras GUTTMAN et. al.

Basic information

Original name

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

Authors

KRENKOVA, Jana (203 Czech Republic), Akos SZEKRENYES (348 Hungary), Zsolt KERESZTESSY (348 Hungary), František FORET (203 Czech Republic, guarantor, belonging to the institution) and Andras GUTTMAN (348 Hungary)

Edition

Journal of Chromatography A, AMSTERDAM, Elsevier, 2013, 0021-9673

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10406 Analytical chemistry

Country of publisher

Netherlands

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 4.258

RIV identification code

RIV/00216224:14740/13:00072093

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.1016/j.chroma.2013.10.087

UT WoS

000328717200007

Keywords in English

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

Abstract

V originále

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.

Links

ED1.1.00/02.0068, research and development project

Name: CEITEC - central european institute of technology

Citovat

KRENKOVA, Jana, Akos SZEKRENYES, Zsolt KERESZTESSY, František FORET and Andras GUTTMAN. Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis. Journal of Chromatography A. AMSTERDAM: Elsevier, 2013, vol. 1322, Dec, p. 54-61. ISSN 0021-9673. Available from: https://dx.doi.org/10.1016/j.chroma.2013.10.087.

@article{1165396,
   author = {Krenkova, Jana and Szekrenyes, Akos and Keresztessy, Zsolt and Foret, František and Guttman, Andras},
   article_location = {AMSTERDAM},
   article_number = {Dec},
   doi = {http://dx.doi.org/10.1016/j.chroma.2013.10.087},
   keywords = {Enzyme microreactor; Oriented immobilization; Monolith; PNGase F; Deglycosylation},
   language = {eng},
   issn = {0021-9673},
   journal = {Journal of Chromatography A},
   title = {Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis},
   volume = {1322},
   year = {2013}
}

TY  - JOUR
ID  - 1165396
AU  - Krenkova, Jana - Szekrenyes, Akos - Keresztessy, Zsolt - Foret, František - Guttman, Andras
PY  - 2013
TI  - Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis
JF  - Journal of Chromatography A
VL  - 1322
IS  - Dec
SP  - 54-61
EP  - 54-61
PB  - Elsevier
SN  - 00219673
KW  - Enzyme microreactor
KW  - Oriented immobilization
KW  - Monolith
KW  - PNGase F
KW  - Deglycosylation
N2  - 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.
ER  -

KRENKOVA, Jana, Akos SZEKRENYES, Zsolt KERESZTESSY, František FORET and Andras GUTTMAN. Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis. \textit{Journal of Chromatography A}. AMSTERDAM: Elsevier, 2013, vol.~1322, Dec, p.~54-61. ISSN~0021-9673. Available from: https://dx.doi.org/10.1016/j.chroma.2013.10.087.

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