earticle

영어

Rapid and reliable quantitative plant N-glycan analysis is highly required to explore complex glycobiological research and develop plant expression systems for factory to produce therapeutic glycoproteins with human compatible glycans. We present here the high throughput quantitative analytical method for plant N-glycan based on DNA sequencing equipment. All steps including peptide N-glycosidase (PNGase) A treatment, glycan preparation and exoglycosidase digestion were optimized for high throughput applications with 96 well format procedures and automatic analysis on DNA sequencer. The glycans of horseradish peroxidase with plant-specific core α(1,3)- fucose can be discriminated by the comparison of glycan profiles obtained by PNGase A and F treatments. The peaks of glycans with (91%) and without (1.2%) α(1, 3)- fucose were easily quantified on a DNA sequencer analysis. In addition, both of them have been shown to harbor bisecting β(1,2)-xylose by simultaneous treatment of α (1,3)-mannosidase and β(1,2)-xylosidase. This technique was also applied to analyze N-glycan of plant-derived anti-rabies monoclonal antibody of which heavy chain was fused to C-terminal Lys-Asp-Glu-Leu (KDEL) sequences for the retention at endoplasmic reticulum. It revealed that most of its N-glycan (more than 80%) belongs to oligomannose type. The identities of glycans generated by DNA sequencer analysis were independently confirmed by mass spectrometry. These results suggest that the DNA sequencer- based method provides quantitative information for plant specific N-glycan analysis in a high throughput manner, which has not been achieved by glycan profiling based on mass spectrometry.