Plant N-glycan profiling of minute amounts of material

• Published in: Analytical biochemistry Vol. 379; no. 1; pp. 66 - 72
• Main Authors: Séveno, Martial, Cabrera, Gleysin, Triguero, Ada, Burel, Carole, Leprince, Jérome, Rihouey, Christophe, Vézina, Louis-Philippe, D’Aoust, Marc-André, Rudd, Pauline M., Royle, L., Dwek, Raymond A., Harvey, David J., Lerouge, Patrice, Cremata, José A., Bardor, Muriel
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2008; Elsevier Masson
• Subjects: Cell Behavior; Cellular Biology; Chemical Sciences; Chromatography, High Pressure Liquid; Glycopeptides; Glycopeptides - chemistry; Glycopeptides - metabolism; Glycoprotein; Life Sciences; Medicago sativa; Medicago sativa - chemistry; Medicinal Chemistry; N-Glycan profiling; Neurobiology; Neurons and Cognition; Nicotiana - chemistry; ortho-Aminobenzoates; ortho-Aminobenzoates - chemistry; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism; Pharmaceutical sciences; Pharmacology; Plant glycosylation; Plant Leaves; Plant Leaves - chemistry; Plants; Plants - chemistry; Polysaccharides; Polysaccharides - analysis; Polysaccharides - chemistry; Polysaccharides - isolation & purification; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Therapeutic protein; Tobacco; N-Glycan profiling; Plant glycosylation; Therapeutic protein; Glycoprotein
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2008.04.034

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 α(1,3)- or α(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.