High-throughput quantitative analysis of plant N-glycan using a DNA sequencer.
Biochem Biophys Res Commun
; 380(2): 223-9, 2009 Mar 06.
Article
em En
| MEDLINE
| ID: mdl-19167352
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Plantas
/
Polissacarídeos
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Proteínas Recombinantes
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Análise de Sequência
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article