Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1216, Issue 15, Pages 3252-3259Publisher
ELSEVIER
DOI: 10.1016/j.chroma.2009.02.036
Keywords
Enzyme reactor; Immobilization; Monolith; Immunoglobulin; PNGase F; Glycomics
Funding
- US Department of Energy [DE-AC02-05CH11231]
- Pfizer Inc.
Ask authors/readers for more resources
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 24 h at 37 degrees C. (C) 2009 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available