title: Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns.
abstract: N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.
authors.affiliation: Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary.
authors.forename: Marton
authors.initials: M
authors.lastname: Szigeti
authors.affiliation: MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary.
authors.forename: Judit
authors.initials: J
authors.lastname: Bondar
authors.affiliation: PhyNexus, Inc., San Jose, CA 95136, United States.
authors.forename: Douglas
authors.initials: D
authors.lastname: Gjerde
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of General Medicine, University of Debrecen, Hungary.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Keresztessy
authors.affiliation: Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary.
authors.forename: Akos
authors.initials: A
authors.lastname: Szekrenyes
authors.affiliation: Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary; MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary. Electronic address: guttman.andras@hlbs.org.
authors.forename: Andras
authors.initials: A
authors.lastname: Guttman
mesh_terms: D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D060748:Fetuins / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry*; D011134:Polysaccharides / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D060748:Fetuins; D006023:Glycoproteins; D007074:Immunoglobulin G; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D012260:Ribonucleases; C029600:ribonuclease B; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Capillary electrophoresis; Enzyme immobilization; N-glycosylation; PNGase F