title: High-throughput quantitative analysis of plant N-glycan using a DNA sequencer.
abstract: High-throughput quantitative analytical method for plant N-glycan has been developed. All steps, including peptide N-glycosidase (PNGase) A treatment, glycan preparation, and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA sequencer. The glycans of horseradish peroxidase with plant-specific core alpha(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) alpha(1,3)-fucose could be readily quantified and shown to harbor bisecting beta(1,2)-xylose via simultaneous treatment with alpha(1,3)-mannosidase and beta(1,2)-xylosidase. This optimized method was successfully applied to analyze N-glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring beta(1,2)-xylose. These results indicate that our DNA sequencer-based method provides quantitative information for plant-specific N-glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry.
authors.affiliation: Integrative Omics Research Center, Korea Research Institute of Bioscience & Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: Kyung Jin
authors.initials: KJ
authors.lastname: Lee
authors.forename: Jin-Hee
authors.initials: JH
authors.lastname: Jung
authors.forename: Jung Mi
authors.initials: JM
authors.lastname: Lee
authors.forename: Yangkang
authors.initials: Y
authors.lastname: So
authors.forename: Ohsuk
authors.initials: O
authors.lastname: Kwon
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
authors.forename: Kisung
authors.initials: K
authors.lastname: Ko
authors.forename: Doo-Byoung
authors.initials: DB
authors.lastname: Oh
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D013058:Mass Spectrometry; D010944:Plants / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011818:Rabies / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017421:Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000295:instrumentation; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D011134:Polysaccharides; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase; D006026:Glycoside Hydrolases