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Anal Biochem. 2008 Aug 1;379(1):66-72. doi: 10.1016/j.ab.2008.04.034. Epub 2008 Apr 25.

Plant N-glycan profiling of minute amounts of material.

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  • 1CNRS-FRE 3090, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan Cédex, France; Medicago Inc., Québec, QC G1V 3V9, Canada.

Abstract

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.

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