High-throughput quantitative analysis of plant N-glycan using a DNA sequencer.
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.
Lee
Kyung Jin
KJ
Integrative Omics Research Center, Korea Research Institute of Bioscience & Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea.
Jung
Jin-Hee
JH
Lee
Jung Mi
JM
So
Yangkang
Y
Kwon
Ohsuk
O
Callewaert
Nico
N
Kang
Hyun Ah
HA
Ko
Kisung
K
Oh
Doo-Byoung
DB
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
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D011134:Polysaccharides; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase; D006026:Glycoside Hydrolases