Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis.
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.
Institute of Analytical Chemistry, v. v. i., Veveri 97, 602 00 Brno, Czech Republic. Electronic address: krenkova@iach.cz.
Jana
J
Krenkova
Akos
A
Szekrenyes
Zsolt
Z
Keresztessy
Frantisek
F
Foret
Andras
A
Guttman
D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D016062:Porosity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D011134:Polysaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Deglycosylation; Enzyme microreactor; Monolith; Oriented immobilization; PNGase F