Multidimensional system enabling deglycosylation of proteins using a capillary reactor with peptide-N-glycosidase F immobilized on a porous polymer monolith and hydrophilic interaction liquid chromatography-mass spectrometry of glycans.
A reactor with immobilized peptide-N-glycosidase F on a monolithic polymer support in a capillary has been developed that allows fast and efficient release of N-linked glycans from immunoglobulin G molecules. Two different monolithic scaffolds based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(butyl methacrylate-co-ethylene dimethacrylate) were prepared. A multistep photografting process was used to reduce non-specific adsorption of proteins and to obtain support containing reactive azlactone functionalities enabling the preparation of highly active immobilized peptide-N-glycosidase F. Performance of these reactors was determined through glycan release from several glycoproteins including ribonuclease B, chicken albumin, and human immunoglobulin G and their detection by matrix-assisted laser desorption-ionization/time-of-flight mass spectrometry. The optimized reactor was integrated into a multidimensional system comprising on-line glycan release and their separation via hydrophilic interaction liquid chromatography followed by electrospray ionization/time-of-flight mass spectrometry detection. Using the optimized monolithic reactor with immobilized peptide-N-glycosidase F, human immunoglobulin G was deglycosylated at room temperature in 5.5 min to an extent similar to that achieved with soluble enzyme after 24h at 37 degrees C.
Krenkova
Jana
J
The Molecular Foundry, EO Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Lacher
Nathan A
NA
Svec
Frantisek
F
D000327:Adsorption; D002853:Chromatography, Liquid / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004867:Equipment Design; D006023:Glycoproteins / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000378:metabolism; D008689:Methacrylates / Q000737:chemistry; D008768:Methylmethacrylates / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis / Q000378:metabolism; D016062:Porosity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D004800:Enzymes, Immobilized; D006023:Glycoproteins; D007074:Immunoglobulin G; D008689:Methacrylates; D008768:Methylmethacrylates; D011134:Polysaccharides; C516546:poly(butyl methacrylate-co-ethylene dimethacrylate); C020347:GMA-EDMA; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase