Rapid Sample Preparation Methodology for Plant N-Glycan Analysis Using Acid-Stable PNGase H+

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• 作者：Ya M. Du ; Tian Xia ; Xiao Q. Gu ; Ting Wang ; Hong Y. Ma ; Josef Voglmeir ; Li Liu
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2015
• 出版时间：December 9, 2015
• 年：2015
• 卷：63
• 期：48
• 页码：10550-10555
• 全文大小：429K
• ISSN：1520-5118

文摘

The quantification of potentially allergenic carbohydrate motifs of plant and insect glycoproteins is increasingly important in biotechnological and agricultural applications as a result of the use of insect cell-based expression systems and transgenic plants. The need to analyze N-glycan moieties in a highly parallel manner inspired us to develop a quick N-glycan analysis method based on a recently discovered bacterial protein N-glycanase (PNGase H⁺). In contrast to the traditionally used PNGase A, which is isolated from almond seeds and only releases N-glycans from proteolytically derived glycopeptides, the herein implemented PNGase H⁺ allows for the release of N-glycans directly from the glycoprotein samples. Because PNGase H⁺ is highly active under acidic conditions, the consecutive fluorescence labeling step using 2-aminobenzamide (2AB) can be directly performed in the same mixture used for the enzymatic deglycosylation step. All sample handling and incubation steps can be performed in less than 4 h and are compatible with microwell-plate sampling, without the need for tedious centrifugation, precipitation, or sample-transfer steps. The versatility of this methodology was evaluated by analyzing glycoproteins derived from various plant sources using ultra-performance liquid chromatography (UPLC) analysis and further demonstrated through the activity analysis of four PNGase H⁺ mutant variants.