Plant N-glycan profiling of minute amounts of material

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• 作者：Martial Sé ; veno ; Gleysin Cabrera ; Ada Triguero ; Carole Burel ; Jé ; rome Leprince ; Christophe Rihouey ; Louis-Philippe Vé ; zina ; Marc-André ; D’ ; Aoust ; Pauline M. Rudd ; L. Royle ; Raymo
• 关键词：Plant glycosylation ; N-Glycan profiling ; Glycoprotein ; Therapeutic protein
• 刊名：Analytical Biochemistry
• 出版年：2008
• 出版时间：1 August 2008
• 年：2008
• 卷：379
• 期：1
• 页码：66-72
• 全文大小：410 K

文摘

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 c=""http://www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0"">(1,3)- or c=""http://www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0"">(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.