title: Enzymatic removal of N-glycans by PNGase F coated magnetic microparticles.
abstract: Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.
authors.affiliation: MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary.
authors.forename: Judit
authors.initials: J
authors.lastname: Bodnar
authors.affiliation: Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.
authors.forename: Akos
authors.initials: A
authors.lastname: Szekrenyes
authors.affiliation: Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.
authors.forename: Marton
authors.initials: M
authors.lastname: Szigeti
authors.affiliation: MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary.|Institute of Analytical Chemistry, Brno, Czech Republic.
authors.forename: Gabor
authors.initials: G
authors.lastname: Jarvas
authors.affiliation: Institute of Analytical Chemistry, Brno, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Krenkova
authors.affiliation: Institute of Analytical Chemistry, Brno, Czech Republic.
authors.forename: Frantisek
authors.initials: F
authors.lastname: Foret
authors.affiliation: MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary.|Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.
authors.forename: Andras
authors.initials: A
authors.lastname: Guttman
mesh_terms: D004586:Electrophoresis; D004800:Enzymes, Immobilized / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008863:Microspheres; D009844:Oligosaccharides / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D006023:Glycoproteins; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011134:Polysaccharides; D012260:Ribonucleases; C029600:ribonuclease B; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Deglycosylation; Enzyme immobilization; Magnetic microparticles; PNGase F